Extraction of lipid from cells and products therefrom

ABSTRACT

The present invention relates to processes for obtaining a lipid from a cell by lysing the cell, contacting the cell with a base and/or salt, and separating the lipid. The present invention is also directed to a lipid prepared by the processes of the present invention. The present invention is also directed to microbial lipids having a particular anisidine value, peroxide value, and/or phosphorus content.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Application No. 61/350,363,filed on Jun. 1, 2010, U.S. Application No. 61/378,923, filed Aug. 31,2010, and U.S. Application No. 61/452,721, filed Mar. 15, 2011, each ofwhich is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to processes for obtaining a lipid from acell by lysing the cell, raising a pH of the cell and/or contacting thecell with a salt, and separating the lipid. The present invention isalso directed to lipids prepared by the processes of the presentinvention. The present invention is also directed to microbial lipidshaving a particular anisidine value, peroxide value, and/or phosphoruscontent.

Background Art

A typical process for obtaining lipids from a microbial cell, such aspolyunsaturated fatty acids, involves growing microorganisms that arecapable of producing the desired lipid in a fermentor, pond orbioreactor, separating the fermentation broth comprising a microbialcell biomass, drying the microbial cell biomass, and separating thelipids by solvent extraction. Steps in the separation can includediluting a fermentation broth with water, centrifuging the dilutedbroth, lysing the microbial cells, and extracting an intracellular lipidfrom the lysed cells by adding a water-immiscible solvent to the mixturein which the lipid is soluble, e.g., hexane.

Another method of extraction to remove a lipid from a microbial cell islysing a cell in a fermentation broth using mechanical force (e.g.,homogenization), enzymatic treatment, or chemical treatment to disruptthe cell walls. Lipid can be extracted from the resulting compositioncomprising lipids, microbial cell biomass, and water using an organicsolvent, e.g., isopropyl alcohol. The lipid can be separatedmechanically from the composition and the alcohol must be removed fromboth the lipid and the aqueous biomass waste stream. See, e.g.,International Pub. Nos. WO 01/76385 and WO 01/76715.

However, industrial scale production of lipids using either of the aboveprocesses requires a large amount of volatile and flammable organicsolvent, thereby creating hazardous operating conditions. The use oforganic solvents in the extraction process can also necessitate using anexplosion-proof lipid recovery system, thereby adding to the cost oflipid recovery. Moreover, use of an organic solvent in extracting lipidfrom a microbial cell can generate an organic solvent waste stream thatrequires a complete solvent recovery system or a proper method ofdisposal, which further increases the overall production cost of lipidextraction. For example, strict limits on volatile organic compound(VOC) emissions require greater manpower and added cost to vessels andother equipment.

Therefore, there is a need for a process for obtaining lipids from acell which does not use an organic solvent. Several processes have beenproposed for separating a lipid from a cell without the use of anorganic solvent. For example, U.S. Pat. No. 6,750,048 discloses anaqueous washing process whereby an emulsion is washed with aqueouswashing solutions until a substantially non-emulsified lipid isobtained. However, in some embodiments, this process requires multiplewashing steps, which require substantial cost and time. U.S. Pat. No.7,431,952 discloses a process whereby lysed cells are centrifuged toremove cell wall debris and then oils are extracted and purified.However, this process provides a crude oil that requires extensivefurther purification. Thus, what is needed is a process that does notutilize a volatile solvent to extract a lipid from a cell, and which canbe performed using readily available equipment and a minimum number ofsteps to provide a highly pure lipid.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to a process for obtaining a lipidfrom a microbial cell composition, the process comprising raising the pHof the cell composition to 8 or above, and separating a lipid from thecell composition, wherein the lipid optionally contains less than 5% byweight or volume of an organic solvent.

In some embodiments, the raising the pH lyses the cell composition. Insome embodiments, the raising the pH demulsifies the cell composition.

In some embodiments, the process comprises adding a salt to the cellcomposition to demulsify the cell composition. In some embodiments, theadding a salt is performed after the raising the pH.

In some embodiments, the process further comprises heating the lysedcell composition to demulsify the cell composition. In some embodiments,the heating is performed after the raising the pH.

In some embodiments, the process further comprises raising the pH of thecell composition a second time to demulsify the cell composition. Insome embodiments, the raising the pH a second time is performed afterthe adding a salt or the heating.

The present invention is also directed to a process for obtaining alipid from a cell, the process comprising lysing a cell to form a lysedcell composition, raising the pH of the lysed cell composition to 8 orabove to demulsify the cell composition, adding a salt to the lysed cellcomposition to demulsify the cell composition, and separating a lipidfrom the demulsified cell composition, wherein the lipid optionallycontains less than 5% by weight or volume of an organic solvent.

The present invention is directed to a process for obtaining a lipidfrom a cell composition, the process comprising raising the pH of thecell composition to 8 or above to lyse the cell composition anddemulsify the cell composition, adding a salt to the cell composition,and separating a lipid from the demulsified cell composition, whereinthe lipid optionally contains less than 5% by weight or volume of anorganic solvent.

The present invention is also directed to a process for obtaining alipid from a cell, the process comprising lysing a cell to form a lysedcell composition, agitating the cell composition to demulsify the cellcomposition, and separating a lipid from the demulsified cellcomposition, wherein the lipid optionally contains less than 5% byweight or volume of an organic solvent.

In some embodiments, the process further comprises heating the lysedcell composition to demulsify the cell composition. In some embodiments,the heating is performed after the adding a salt.

In some embodiments, the process further comprises agitating the lysedcell composition to demulsify the cell composition. In some embodiments,the agitating is for 5 minutes to 96 hours.

In some embodiments, the agitating comprises agitating the cellcomposition with an impeller having a tip speed of 350 centimeters persecond to 900 centimeters per second.

In some embodiments, the process further comprises raising the pH of thelysed cell composition to demulsify the cell composition. In someembodiments, raising the pH of the lysed cell composition to demulsifythe cell composition comprises adding a base. In some embodiments, asecond base is added after the adding of a salt or the heating.

In some embodiments, the heating is for 10 minutes to 96 hours.

In some embodiments, the cell composition is heated to a temperature of60° C. to 100° C. In some embodiments, the cell composition is heated toa temperature of 90° C. to 100° C.

In some embodiments, raising the pH comprises adding a base. In someembodiments, the base has a pK_(b) of 1 to 12.

In some embodiments, the separating a lipid occurs at a temperature of10° C. to 100° C.

In some embodiments, the process comprises agitating the lysed cellcomposition by stirring, mixing, blending, shaking, vibrating, or acombination thereof. In some embodiments, the process comprisesagitating the lysed cell composition at 0.1 hp/1000 gal to 10 hp/1000gal of lysed cell composition. In some embodiments, the processcomprises agitating the lysed cell composition with an agitator havingan impeller tip speed of 200 ft/min to 1,000 ft/min.

In some embodiments, the lysing comprises mechanical treatment, physicaltreatment, chemical treatment, enzymatic treatment, or a combinationthereof. In some embodiments, the mechanical treatment ishomogenization.

In some embodiments, the salt is added in an amount of 0.1% to 20% byweight of the lysed cell composition. In some embodiments, the salt isadded to the lysed cell composition in an amount of 0.5% to 15% byweight of the lysed cell composition. In some embodiments, the salt isadded to the lysed cell composition in an amount of 2% to 10% by weightof the lysed cell composition.

In some embodiments, the salt is selected from the group consisting of:alkali metal salts, alkali earth metal salts, sulfate salts, andcombinations thereof. In some embodiments, the salt is sodium chloride.In some embodiments, the salt is sodium sulfate.

In some embodiments, the separating comprises centrifuging. In someembodiments, the separating comprises centrifuging at a temperature of30° C. to 90° C.

In some embodiments, the process provides a lipid comprising at least50% by weight triglyceride.

In some embodiments, the process provides a lipid having an anisidinevalue of 26 or less, 25 or less, 20 or less, 15 or less, 10 or less, 5or less, 2 or less, or 1 or less.

In some embodiments, the process provides a lipid having a peroxidevalue of 5 or less, 4.5 or less, 4 or less, 3.5 or less, 3 or less, 2.5or less, 2 or less, 1.5 or less, 1 or less, 0.5 or less, 0.2 or less, or0.1 or less.

In some embodiments, the process provides a lipid having a phosphoruscontent of 100 ppm or less, 95 ppm or less, 90 ppm or less, 85 ppm orless, 80 ppm or less, 75 ppm or less, 70 ppm or less, 65 ppm or less, 60ppm or less, 55 ppm or less, 50 ppm or less, 45 ppm or less, 40 ppm orless, 35 ppm or less, 30 ppm or less, 25 ppm or less, 20 ppm or less, 15ppm or less, 10 ppm or less, 5 ppm or less, 4 ppm or less, 3 ppm orless, 2 ppm or less, or 1 ppm or less.

In some embodiments, the process provides a lipid having at least 10%,at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, or at least 50% by weight of a desiredpolyunsaturated fatty acid (PUFA). In some embodiments, the processprovides a lipid having at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 35%, at least 40%, at least 45%, or atleast 50% by weight of docosahexaenoic acid (“DHA”), and/or at least10%, at least 15%, or at least 20% by weight of docosapentaenoic acid(“DPA n-6”), and/or at least 10%, at least 15%, or at least 20% byweight of eicosapentaenoic acid (“EPA”), and/or at least 10%, at least15%, at least 20%, at least 25%, at least 30%, at least 35%, at least40%, at least 45%, or at least 50% by weight of arachidonic acid(“ARA”).

In some embodiments, the cell is a microbial cell. In some embodiments,the process comprises concentrating a fermentation broth comprising themicrobial cell.

In some embodiments, the cell is an oilseed. In some embodiments, theoilseed is selected from the group consisting of sunflower seeds, canolaseeds, rapeseeds, linseeds, castor oil seeds, coriander seeds, calendulaseeds, and genetically modified variants thereof.

In some embodiments, the process comprises washing the cell or cellcomposition.

In some embodiments, the process comprises pasteurizing the cell or cellcomposition.

In some embodiments, the process comprises concentrating the lysed cellcomposition.

In some embodiments, the process comprises refining the lipid. In someembodiments, the refining is selected from the group consisting of:caustic refining, degumming, acid treatment, alkali treatment, cooling,heating, bleaching, deodorizing, deacidification, and combinationsthereof.

In some embodiments, the process comprises harvesting the lipid, whereinthe harvesting comprises pumping the lipid without agitation.

The present invention is also directed to a lipid obtained by any of theprocesses of the present invention.

In some embodiments, the lipid comprises one or more polyunsaturatedfatty acids. In some embodiments, the lipid has at least 10%, at least15%, at least 200%, at least 25%, at least 30%, at least 35%, at least40%, at least 45%, or at least 50% by weight of a desired PUFA. In someembodiments, the lipid has at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 35%, at least 40%, at least 450%, orat least 50% by weight of DHA, and/or at least 10%, at least 15%, or atleast 20% by weight of DPA n-6, and/or at least 10%, at least 15%, or atleast 20% by weight of EPA, and/or at least 100%, at least 15%, at least20%, at least 25%, at least 30%, at least 35%, at least 40%, at least45%, or at least 50% by weight of ARA.

In some embodiments, the lipid has an overall aroma intensity of 3 orless. In some embodiments, the lipid has an overall aromatic intensityof 2 or less.

In some embodiments, the lipid comprises a triacylglycerol fraction ofat least 10% by weight, wherein at least 12% by weight of the fattyacids in the triacylglycerol fraction is eicosapentaenoic acid, whereinat least 25% by weight of the fatty acids in the triacylglycerolfraction is docosahexaenoic acid, and wherein less than 5% by weight ofthe fatty acids in the triacylglycerol fraction is arachidonic acid.

In some embodiments, the lipid comprises at least 20% by weighteicosapentaenoic acid and less than 5% by weight each of arachidonicacid, docosapentaenoic acid n-6, oleic acid, linoleic acid, linolenicacid, eicosenoic acid, erucic acid, and stearidonic acid.

In some embodiments, the lipid has an anisidine value of 26 or less, 25or less, 20 or less, 15 or less, 10 or less, 5 or less, 2 or less, or 1or less, and/or a peroxide value of 5 or less, 4.5 or less, 4 or less,3.5 or less, 3 or less, 2.5 or less, 2 or less, 1.5 or less, 1 or less,0.5 or less, 0.2 or less, or 0.1 or less, and/or a phosphorus content of100 ppm or less, 95 ppm or less, 90 ppm or less, 85 ppm or less, 80 ppmor less, 75 ppm or less, 70 ppm or less, 65 ppm or less, 60 ppm or less,55 ppm or less, 50 ppm or less, 45 ppm or less, 40 ppm or less, 35 ppmor less, 30 ppm or less, 25 ppm or less, 20 ppm or less, 15 ppm or less,10 ppm or less, 5 ppm or less, 4 ppm or less, 3 ppm or less, 2 ppm orless, or 1 ppm or less.

In some embodiments, the lipid is a crude lipid. In some embodiments,the crude lipid optionally has less than 5% by weight or volume of anorganic solvent.

The present invention is also directed to a crude microbial lipid havingan anisidine value of 26 or less, a peroxide value of 5 or less, aphosphorus content of 100 ppm or less, and optionally less than 5% byweight or volume of an organic solvent.

In some embodiments, the crude microbial lipid has an anisidine value of26 or less, 25 or less, 20 or less, 15 or less, 10 or less, 5 or less, 2or less, or 1 or less, and/or a peroxide value of 5 or less, 4.5 orless, 4 or less, 3.5 or less, 3 or less, 2.5 or less, 2 or less, 1.5 orless, 1 or less, 0.5 or less, 0.2 or less, or 0.1 or less, and/or aphosphorus content of 100 ppm or less, 95 ppm or less, 90 ppm or less,85 ppm or less, 80 ppm or less, 75 ppm or less, 70 ppm or less, 65 ppmor less, 60 ppm or less, 55 ppm or less, 50 ppm or less, 45 ppm or less,40 ppm or less, 35 ppm or less, 30 ppm or less, 25 ppm or less, 20 ppmor less, 15 ppm or less, 10 ppm or less, 5 ppm or less, 4 ppm or less, 3ppm or less, 2 ppm or less, or 1 ppm or less.

In some embodiments, the crude microbial lipid has at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, or at least 50% by weight of a desired PUFA. Insome embodiments, the crude microbial lipid has at least 10%, at least15%, at least 20%, at least 25%, at least 30%, at least 35%, at least40%, at least 45%, or at least 50% by weight of DHA, and/or at least10%, at least 15%, or at least 20% by weight of DPA n-6, and/or at least10%, at least 15%, or at least 20% by weight of EPA, and/or at least10%, at least 15%, at least 20%, at least 25%, at least 30%, at least35%, at least 40%, at least 45%, or at least 50% by weight of ARA.

The present invention is also directed to an extracted microbial lipidcomprising a triglyceride fraction of at least 70% by weight, whereinthe docosahexaenoic acid content of the triglyceride fraction is atleast 500 by weight, wherein the docosapentaenoic acid n-6 content ofthe triglyceride fraction is from at least 0.5% by weight to 6% byweight, and wherein the oil has an anisidine value of 26 or less.

The present invention is also directed to an extracted microbial lipidcomprising a triglyceride fraction of at least 70% by weight, whereinthe docosahexaenoic acid content of the triglyceride fraction is atleast 40% by weight, wherein the docosapentaenoic acid n-6 content ofthe triglyceride fraction is from at least 0.5% by weight to 6% byweight, wherein the ratio of docosahexaenoic acid to docosapentaenoicacid n-6 is greater than 6:1, and wherein the oil has an anisidine valueof 26 or less.

The present invention is also directed to an extracted microbial lipidcomprising a triglyceride fraction of at least about 70% by weight,wherein the docosahexaenoic acid content of the triglyceride fraction isat least 60% by weight and wherein the oil has an anisidine value of 26or less.

In some embodiments, the extracted lipid has an anisidine value of 26 orless, 25 or less, 20 or less, 15 or less, 10 or less, 5 or less, 2 orless, or 1 or less, and/or a peroxide value of 5 or less, 4.5 or less, 4or less, 3.5 or less, 3 or less, 2.5 or less, 2 or less, 1.5 or less, 1or less, 0.5 or less, 0.2 or less, or 0.1 or less, and/or a phosphoruscontent of 100 ppm or less, 95 ppm or less, 90 ppm or less, 85 ppm orless, 80 ppm or less, 75 ppm or less, 70 ppm or less, 65 ppm or less, 60ppm or less, 55 ppm or less, 50 ppm or less, 45 ppm or less, 40 ppm orless, 35 ppm or less, 30 ppm or less, 25 ppm or less, 20 ppm or less, 15ppm or less, 10 ppm or less, 5 ppm or less, 4 ppm or less, 3 ppm orless, 2 ppm or less, or 1 ppm or less.

In some embodiments, the extracted microbial lipid is a crude lipid or acrude oil. In some embodiments, the crude lipid optionally has less than5% by weight or volume of an organic solvent.

The present invention is also directed to a process for obtaining alipid, the process comprising refining a crude lipid of the presentinvention. In some embodiments, the refining is selected from the groupconsisting of: caustic refining, degumming, acid treatment, alkalitreatment, cooling, heating, bleaching, deodorizing, deacidification,and combinations thereof.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

The accompanying drawings, which are incorporated herein and form a partof the specification, illustrate the present invention and, togetherwith the description, further serve to explain the principles of theinvention and to enable a person skilled in the pertinent art to makeand use the invention.

FIGS. 1-4 provide schematic flow charts describing processes of thepresent invention.

FIG. 5 is a graph providing the electron paramagnetic resonance (EPR)over time of lysed cells compositions at various pHs.

The present invention will now be described with reference to theaccompanying drawings. In the drawings, like reference numbers indicateidentical or functionally similar elements. Additionally, the left-mostdigit(s) of a reference number can identify the drawing in which thereference number first appears.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a process for obtaining a lipidfrom a microbial cell composition, the process comprising raising the pHof the cell composition to 8 or above and separating a lipid from thecell composition, wherein the lipid optionally contains less than 5% byweight or volume of an organic solvent. In some embodiments, the processfurther comprises one or more of adding a salt to the cell compositionto demulsify the cell composition, heating the cell to demulsify thecell composition, agitating the cell composition to demulsify the cellcomposition, and raising the pH of the cell composition a second time todemulsify the cell composition.

The present invention is also directed to a process for obtaining alipid from a cell, the process comprising lysing a cell to form a lysedcell composition, raising the pH of the lysed cell composition to 8 orabove to demulsify the cell composition, adding a salt to the lysed cellcomposition to demulsify the cell composition, and separating a lipidfrom the demulsified cell composition, wherein the lipid optionallycontains less than 5% by weight or volume of an organic solvent. Thecell can be a microbial cell or an oilseed cell. In some embodiments,the process further comprises one or more of: heating the lysed cellcomposition to demulsify the cell composition, agitating the lysed cellcomposition to demulsify the cell composition, and raising the pH of thelysed cell composition a second time to demulsify the cell composition.

The present invention is directed to a process for obtaining a lipidfrom a cell composition, the process comprising raising the pH of thecell composition to 8 or above to lyse the cell composition anddemulsify the cell composition, adding a salt to the cell composition,and separating a lipid from the demulsified cell composition, whereinthe lipid optionally contains less than 5% by weight or volume of anorganic solvent. In some embodiments, the process further comprises oneor more of heating the cell composition to demulsify the cellcomposition, agitating the cell composition to demulsify the cellcomposition, and raising the pH of the cell composition a second time todemulsify the cell composition.

The present invention is directed to a process for obtaining a lipidfrom a microbial cell, the process comprising lysing a microbial cell toform a lysed cell composition, adding a base to the lysed cellcomposition to demulsify the cell composition, and separating a lipidfrom the demulsified cell composition, wherein the lipid optionallycontains less than 5% by weight or volume of an organic solvent. In someembodiments, the process further comprises one or more of: adding a saltto the lysed cell composition to demulsify the cell composition, heatingthe lysed cell composition to demulsify the cell composition, agitatingthe lysed cell composition to demulsify the cell composition, and addinga second base to the lysed cell composition to demulsify the cellcomposition.

The present invention is also directed to a process for obtaining alipid from a cell, the process comprising lysing a cell to form a lysedcell composition, adding a base to the lysed cell composition todemulsify the cell composition, adding a salt to the lysed cellcomposition to demulsify the cell composition, and separating a lipidfrom the demulsified cell composition, wherein the lipid optionallycontains less than 5% by weight or volume of an organic solvent. Thecell can be a microbial cell or an oilseed cell. In some embodiments,the process further comprises one or more of: heating the lysed cellcomposition to demulsify the cell composition, agitating the lysed cellcomposition to demulsify the cell composition, and adding a second baseto the lysed cell composition to demulsify the cell composition.

The present invention is also directed to a process for obtaining alipid from a cell, the process comprising lysing a cell to form a lysedcell composition, agitating the cell composition to demulsify the cellcomposition, and separating a lipid from the demulsified cellcomposition, wherein the lipid optionally contains less than 5% byweight or volume of an organic solvent.

The present invention is also directed to a lipid obtained by any of theprocesses of the present invention.

The present invention is also directed to an extraction process forobtaining a lipid from a cell, the process comprising lysing the cell toform a lysed cell composition, contacting the lysed cell compositionwith a first base, contacting the lysed cell composition with a salt,heating the lysed cell composition for 5 minutes to 96 hours, contactingthe lysed cell composition with a second base, and separating a lipidfrom the lysed cell composition at a temperature of 10° C. to 100° C.

The present invention is also directed to an extraction process forobtaining a lipid from a cell, the process comprising lysing the cell toform a lysed cell composition, contacting the lysed cell compositionwith a salt, and agitating the lysed cell composition for 5 minutes to96 hours to provide a treated lysed cell composition, and separating alipid from the treated lysed cell composition at a temperature of 10° C.to 100° C.

The present invention is also directed to an extraction process forobtaining a lipid from a cell, the process comprising lysing the cell toform a lysed cell composition, contacting the lysed cell compositionwith a salt, and separating a lipid from the lysed cell composition at atemperature of 10° C. to 100° C.

In some embodiments, the base or second base have a pK_(b) of 1 to 12.In some embodiments, the base or second base have a pK_(b) of 3 to 5.

In some embodiments, a process comprises agitating the lysed cellcomposition for 5 minutes to 96 hours, 10 minutes to 96 hours, 10minutes to 4 hours, 12 hours to 84 hours, or 24 hours to 72 hours.

In some embodiments, the process comprises agitating the lysed cellcomposition by stirring, mixing, blending, shaking, vibrating, or acombination thereof. In some embodiments, the process comprisesagitating the lysed cell composition at 0.1 hp/1000 gal to 10 hp/1000gal of lysed cell composition. In some embodiments, the processcomprises agitating the lysed cell composition with an agitator havingan impeller tip speed of 200 ft/min to 1000 ft/min.

In some embodiments, lysing comprises a process selected from:mechanically treating, physically treating, chemically treating,enzymatically treating, or a combination thereof.

In some embodiments, the lysed cell composition is contacted with a saltin an amount of 0.1% to 20% by weight, 0.5% to 15% by weight, or 2% to10% by weight of the lysed cell composition.

In some embodiments, the salt is selected from the group consisting of:alkali metal salts, alkali earth metal salts, sulfate salts andcombinations thereof. In some embodiments, the salt is sodium chloride.In some embodiments, the salt is sodium sulfate.

In some embodiments, the process comprises heating the lysed cellcomposition for 5 minutes to 96 hours, 10 minutes to 4 hours, 12 hoursto 84 hours, or 24 hours to 72 hours.

In some embodiments, the separating comprises centrifuging. In someembodiments, the separating comprises centrifuging at a temperature of10° C. to 100° C.

In some embodiments, the process comprises prior to the lysing: washing,centrifuging, evaporating, or a combination thereof, a broth thatincludes the cell.

In some embodiments, the process provides a lipid having an anisidinevalue of 15 or less. In some embodiments, the process provides a lipidcomprising at least 50% by weight triglycerides.

In some embodiments, the process does not add an organic solvent to thelysed cell composition. Organic solvents include polar solvents,non-polar solvents, water-miscible solvents, water-immiscible solvents,and combinations thereof.

In some embodiments, the process comprises concentrating a brothcomprising a cell. In some embodiments, the process comprisesconcentrating the lysed cell composition.

The present invention is also directed to the lipid prepared by aprocess described herein. In some embodiments, the lipid comprises oneor more polyunsaturated fatty acids. In some embodiments, the lipid hasat least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, at least 45%, or at least 50% by weight of adesired PUFA. In some embodiments, the lipid has at least 10%, at least15%, at least 20%, at least 25%, at least 30%, at least 35%, at least40%, at least 45%, or at least 50% by weight of DHA, and/or at least10%, at least 15%, or at least 20% by weight of DPA n-6, and/or at least10%, at least 15%, or at least 20% by weight of EPA, and/or at least10%, at least 15%, at least 20%, at least 25%, at least 30%, at least35%, at least 40%, at least 45%, or at least 50% by weight of ARA. Insome embodiments, the lipid has an anisidine value of 26 or less, 25 orless, 20 or less, 15 or less, 10 or less, 5 or less, 2 or less, or 1 orless, and/or a peroxide value of 5 or less, 4.5 or less, 4 or less, 3.5or less, 3 or less, 2.5 or less, 2 or less, 1.5 or less, 1 or less, 0.5or less, 0.2 or less, or 0.1 or less, and/or a phosphorus content of 100ppm or less, 95 ppm or less, 90 ppm or less, 85 ppm or less, 80 ppm orless, 75 ppm or less, 70 ppm or less, 65 ppm or less, 60 ppm or less, 55ppm or less, 50 ppm or less, 45 ppm or less, 40 ppm or less, 35 ppm orless, 30 ppm or less, 25 ppm or less, 20 ppm or less, 15 ppm or less, 10ppm or less, 5 ppm or less, 4 ppm or less, 3 ppm or less, 2 ppm or less,or 1 ppm or less.

In some embodiments, the lipid comprises a triacylglycerol fraction ofat least 10% by weight, wherein at least 12% by weight of the fattyacids in the triacylglycerol fraction is eicosapentaenoic acid, whereinat least 25% by weight of the fatty acids in the triacylglycerolfraction is docosahexaenoic acid, and wherein less than 5% by weight ofthe fatty acids in the triacylglycerol fraction is arachidonic acid. Insome embodiments, the lipid has an anisidine value of 26 or less, 25 orless, 20 or less, 15 or less, 10 or less, 5 or less, 2 or less, or 1 orless, and/or a peroxide value of 5 or less, 4.5 or less, 4 or less, 3.5or less, 3 or less, 2.5 or less, 2 or less, 1.5 or less, 1 or less, 0.5or less, 0.2 or less, or 0.1 or less, and/or a phosphorus content of 100ppm or less, 95 ppm or less, 90 ppm or less, 85 ppm or less, 80 ppm orless, 75 ppm or less, 70 ppm or less, 65 ppm or less, 60 ppm or less, 55ppm or less, 50 ppm or less, 45 ppm or less, 40 ppm or less, 35 ppm orless, 30 ppm or less, 25 ppm or less, 20 ppm or less, 15 ppm or less, 10ppm or less, 5 ppm or less, 4 ppm or less, 3 ppm or less, 2 ppm or less,or 1 ppm or less. In some embodiments, the lipid is a crude lipid.

In some embodiments, the lipid comprises at least 20% by weighteicosapentaenoic acid and less than 5% by weight each of arachidonicacid, docosapentaenoic acid n-6, oleic acid, linoleic acid, linolenicacid, eicosenoic acid, erucic acid, and stearidonic acid. In someembodiments, the lipid has an anisidine value of 26 or less, 25 or less,20 or less, 15 or less, 10 or less, 5 or less, 2 or less, or 1 or less,and/or a peroxide value of 5 or less, 4.5 or less, 4 or less, 3.5 orless, 3 or less, 2.5 or less, 2 or less, 1.5 or less, 1 or less, 0.5 orless, 0.2 or less, or 0.1 or less, and/or a phosphorus content of 100ppm or less, 95 ppm or less, 90 ppm or less, 85 ppm or less, 80 ppm orless, 75 ppm or less, 70 ppm or less, 65 ppm or less, 60 ppm or less, 55ppm or less, 50 ppm or less, 45 ppm or less, 40 ppm or less, 35 ppm orless, 30 ppm or less, 25 ppm or less, 20 ppm or less, 15 ppm or less, 10ppm or less, 5 ppm or less, 4 ppm or less, 3 ppm or less, 2 ppm or less,or 1 ppm or less. In some embodiments, the lipid is a crude oil.

The present invention is also directed to a crude microbial lipid havingan anisidine value of 26 or less, a peroxide value of 5 or less, aphosphorus content of 100 ppm or less, and optionally less than 5% byweight or volume of an organic solvent. In some embodiments, the crudemicrobial lipid has an anisidine value of 26 or less, 25 or less, 20 orless, 15 or less, 10 or less, 5 or less, 2 or less or 1 or less, and/ora peroxide value of 5 or less, 4.5 or less, 4 or less, 3.5 or less, 3 orless, 2.5 or less, 2 or less, 1.5 or less, 1 or less, 0.5 or less, 0.2or less, or 0.1 or less, and/or a phosphorus content of 100 ppm or less,95 ppm or less, 90 ppm or less, 85 ppm or less, 80 ppm or less, 75 ppmor less, 70 ppm or less, 65 ppm or less, 60 ppm or less, 55 ppm or less,50 ppm or less, 45 ppm or less, 40 ppm or less, 35 ppm or less, 30 ppmor less, 25 ppm or less, 20 ppm or less, 15 ppm or less, 10 ppm or less,5 ppm or less, 4 ppm or less, 3 ppm or less, 2 ppm or less, or 1 ppm orless. In some embodiments, the crude microbial lipid has at least 10%,at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, or at least 50% by weight of a desired PUFA. Insome embodiments, the crude microbial lipid has at least 10%, at least15%, at least 20%, at least 25%, at least 30%, at least 35%, at least40%, at least 45%, or at least 50% by weight of DHA, and/or at least10%, at least 15%, or at least 20% by weight of DPA n-6, and/or at least10%, at least 15%, or at least 20% by weight of EPA, and/or at least10%, at least 15%, at least 20%, at least 25%, at least 30%, at least35%, at least 40%, at least 45%, or at least 50% by weight of ARA.

The present invention is also directed to an extracted microbial lipidcomprising a triglyceride fraction of at least 70% by weight, whereinthe docosahexaenoic acid content of the triglyceride fraction is atleast 50% by weight, wherein the docosapentaenoic acid n-6 content ofthe triglyceride fraction is from at least 0.5% by weight to 6% byweight, and wherein the oil has an anisidine value of 26 or less. Insome embodiments, the extracted lipid has an anisidine value of 26 orless, 25 or less, 20 or less, 15 or less, 10 or less, 5 or less, 2 orless, or 1 or less, and/or a peroxide value of 5 or less, 4.5 or less, 4or less, 3.5 or less, 3 or less, 2.5 or less, 2 or less, 1.5 or less, 1or less, 0.5 or less, 0.2 or less, or 0.1 or less, and/or a phosphoruscontent of 100 ppm or less, 95 ppm or less, 90 ppm or less, 85 ppm orless, 80 ppm or less, 75 ppm or less, 70 ppm or less, 65 ppm or less, 60ppm or less, 55 ppm or less, 50 ppm or less, 45 ppm or less, 40 ppm orless, 35 ppm or less, 30 ppm or less, 25 ppm or less, 20 ppm or less, 15ppm or less, 10 ppm or less, 5 ppm or less, 4 ppm or less, 3 ppm orless, 2 ppm or less, or 1 ppm or less. In some embodiments, theextracted lipid is a crude lipid.

The present invention is also directed to an extracted microbial lipidcomprising a triglyceride fraction of at least 70% by weight, whereinthe docosahexaenoic acid content of the triglyceride fraction is atleast 40% by weight, wherein the docosapentaenoic acid n-6 content ofthe triglyceride fraction is from at least 0.5% by weight to 6% byweight, wherein the ratio of docosahexaenoic acid to docosapentaenoicacid n-6 is greater than 6:1, and wherein the oil has an anisidine valueof 26 or less. In some embodiments, the extracted lipid has an anisidinevalue of 26 or less, 25 or less, 20 or less, 15 or less, 10 or less, 5or less, 2 or less, or 1 or less, and/or a peroxide value of 5 or less,4.5 or less, 4 or less, 3.5 or less, 3 or less, 2.5 or less, 2 or less,1.5 or less, 1 or less, 0.5 or less, 0.2 or less, or 0.1 or less, and/ora phosphorus content of 100 ppm or less, 95 ppm or less, 90 ppm or less,85 ppm or less, 80 ppm or less, 75 ppm or less, 70 ppm or less, 65 ppmor less, 60 ppm or less, 55 ppm or less, 50 ppm or less, 45 ppm or less,40 ppm or less, 35 ppm or less, 30 ppm or less, 25 ppm or less, 20 ppmor less, 15 ppm or less, 10 ppm or less, 5 ppm or less, 4 ppm or less, 3ppm or less, 2 ppm or less, or 1 ppm or less. In some embodiments, theextracted lipid is a crude lipid.

The present invention is also directed to an extracted microbial lipidcomprising a triglyceride fraction of at least about 70% by weight,wherein the docosahexaenoic acid content of the triglyceride fraction isat least 60/o by weight and wherein the oil has an anisidine value of 26or less. In some embodiments, the extracted lipid has an anisidine valueof 26 or less, 25 or less, 20 or less, 15 or less, 10 or less, 5 orless, 2 or less, or 1 or less, and/or a peroxide value of 5 or less, 4.5or less, 4 or less, 3.5 or less, 3 or less, 2.5 or less, 2 or less, 1.5or less, 1 or less, 0.5 or less, 0.2 or less, or 0.1 or less, and/or aphosphorus content of 100 ppm or less, 95 ppm or less, 90 ppm or less,85 ppm or less, 80 ppm or less, 75 ppm or less, 70 ppm or less, 65 ppmor less, 60 ppm or less, 55 ppm or less, 50 ppm or less, 45 ppm or less,40 ppm or less, 35 ppm or less, 30 ppm or less, 25 ppm or less, 20 ppmor less, 15 ppm or less, 10 ppm or less, 5 ppm or less, 4 ppm or less, 3ppm or less, 2 ppm or less, or 1 ppm or less. In some embodiments, theextracted lipid is a crude lipid.

The present invention is also directed to a crude lipid extracted from amicroorganism of the species Crypthecodinium cohnii, having a phosphoruscontent of 100 ppm or less. In some embodiments, the crude lipid has ananisidine value of 26 or less, 25 or less, 20 or less, 15 or less, 10 orless, 5 or less, 2 or less, or 1 or less, and/or a peroxide value of 5or less, 4.5 or less, 4 or less, 3.5 or less, 3 or less, 2.5 or less, 2or less, 1.5 or less, 1 or less, 0.5 or less, 0.2 or less, or 0.1 orless, and/or a phosphorus content of 100 ppm or less, 95 ppm or less, 90ppm or less, 85 ppm or less, 80 ppm or less, 75 ppm or less, 70 ppm orless, 65 ppm or less, 60 ppm or less, 55 ppm or less, 50 ppm or less, 45ppm or less, 40 ppm or less, 35 ppm or less, 30 ppm or less, 25 ppm orless, 20 ppm or less, 15 ppm or less, 10 ppm or less, 5 ppm or less, 4ppm or less, 3 ppm or less, 2 ppm or less, or 1 ppm or less.

The present invention is also directed to a process for obtaining alipid, the process comprising refining a crude lipid of the presentinvention. In some embodiments, the refining is selected from the groupconsisting of: caustic refining, degumming, acid treatment, alkalitreatment, cooling, heating, bleaching, deodorizing, deacidification,and combinations thereof.

Overview

Generally, the processes of the present invention do not utilize anorganic solvent in order to extract or otherwise separate a lipid. Thus,in some embodiments, an organic solvent is not added to a cell brothcomprising plant material or fermentation broth comprising a microbialcell, is not added to a cell composition, is not added to a lysed cellcomposition, or is not added to a lipid during a process of the presentinvention in an amount or concentration sufficient to extract a lipid.In some embodiments, an organic solvent can be added to a cellcomposition, a lysed cell composition, or a demulsified cellcomposition. In such embodiments, the organic solvent is added in aconcentration less than 5%, less than 4%, less than 3%, less than 2%,less than 1%, less than 0.5%, less than 0.1%, or less than 0.05% byvolume. As used herein, an “organic solvent” refers to a solvent thatincludes at least one carbon atom. As used herein, “solvent” refers toan agent that is hydrophobic or lipophilic, and is not a lipid. As usedherein, “hydrophobic” refers to an agent that is repelled from a mass ofwater. As used herein, “lipophilic” refers to an agent that dissolveslipids. Organic solvents that are not used in a process of the presentinvention include, but are not limited to, polar solvents, non-polarsolvents, water-miscible solvents, water-immiscible solvents, andcombinations thereof. Non-limiting examples of organic solvents includesubstituted and unsubstituted C₄-C₈ alkyls (e.g., hexane and the like),C₅-C₁₂ cylcolalkyls, C₄-C₁₂ alkenes, C₁-C₈ alcohols (e.g., iso-propanoland the like), C₁-C₈ aldehydes, C₄—C ethers, C₁-C₈ esters, C₆-C₁₂ aryls,C₁-C₈ amides, C₅-C₁₂ heteroaryls, and combinations thereof. An organicsolvent as defined herein can be optionally added to a lysed cellcomposition, for example, as a component of a base and/or a salt forcontacting with the lysed cell composition. However, in such embodimentsthe organic solvent is present in a concentration such that the lipid isnot substantially extracted from the cell composition, lysed cellcomposition, or demulsified cell composition by the solvent (i.e., in aconcentration of less than 5%, less than 4%, less than 3%, less than 2%,less than 1%, less than 0.5%, less than 0.1%, or less than 0.05% byvolume or weight).

In some embodiments, a process of the present invention does not includewashing, e.g., with water, or the process reduces the number of washingsof, a lysed cell composition or a demulsified cell composition.“Washing” refers to a process of diluting a composition with, e.g.,water or buffer and removing the water or buffer, e.g., bycentrifugation. Washing a cell composition can decrease the overallyield of a lipid obtained from a cell. In the present invention, thewashing can be decreased by 1 time, 2 times, 3 times or more.

Definitions

As used herein, “lipid” or “oil” refers to one or more fatty acids(including free fatty acids and esters of fatty acids), phospholipids,triacylglycerols (i.e. triglycerides), diacylglycerides,monoacylglycerides, lysophospholipids, soaps, phosphatides, waxes,sterols and sterol esters, carotenoids, xanthophylls, hydrocarbons, andother lipids known to one of ordinary skill in the art. Lipids includepolar lipids and neutral lipids.

As used herein, “polar lipid” refers to lipids that contain a polargroup and are more readily soluble in polar solvents. Polar lipidsinclude phospholipids. As used herein, “phospholipid” refers to lipidshaving a phosphate group. As used herein, “neutral lipid” refers tolipids that do not contain areas of polarity and are more readilysoluble in non-polar solvents. Neutral lipids include triacylglycerols(TAG).

Fatty acids are classified based on the length and saturationcharacteristics of the carbon chain. Fatty acids are termed short chain,medium chain, or long chain fatty acids based on the number of carbonspresent in the chain. Fatty acids are termed saturated fatty acids whenno double bonds are present between the carbon atoms, and are termedunsaturated fatty acids when double bonds are present. Unsaturated longchain fatty acids are monounsaturated when only one double bond ispresent and are polyunsaturated when more than one double bond ispresent.

Fatty acids present in the lipid can have 4 to 28 carbon atoms. In someembodiments, a lipid comprises one or more polyunsaturated fatty acids.Polyunsaturated fatty acids (PUFAs) are classified based on the positionof the first double bond from the methyl end of the fatty acid: omega-3(n-3) fatty acids contain a first double bond at the third carbon, whileomega-6 (n-6) fatty acids contain a first double bond at the sixthcarbon. For example, docosahexaenoic acid (“DHA”) is an omega-3 longchain polyunsaturated fatty acid (LC-PUFA) with a chain length of 22carbons and 6 double bonds, often designated as “22:6 n-3.” For thepurposes of this application, long chain polyunsaturated fatty acids(LC-PUFAs) are defined as fatty acids of 18 and more carbon chainlength, and are preferably fatty acids of 20 or more carbon chainlength, containing 3 or more double bonds. LC-PUFAs of the omega-6series include, but are not limited to, di-homo-gammalinoleic acid(C20:3n-6), arachidonic acid (C20:4n-6) (“ARA”), docosatetraenoic acidor adrenic acid (C22:4n-6), and docosapentaenoic acid (C22:5n-6) (“DPAn-6”). The LC-PUFAs of the omega-3 series include, but are not limitedto, eicosatrienoic acid (C20:3n-3), eicosatetraenoic acid (C20:4n-3),eicosapentaenoic acid (C20:5n-3) (“EPA”), docosapentaenoic acid(C22:5n-3), and docosahexaenoic acid (C22:6n-3). The LC-PUFAs alsoinclude fatty acids with greater than 22 carbons and 4 or more doublebonds including, but not limited to, C24:6(n-3) and C28:8(n-3).

The terms “fatty acid,” “polyunsaturated fatty acid,” and “PUFA” includenot only the free fatty acid form, but other forms as well, such as thetriacylglycerol (TAG) form, the phospholipid (PL) form and otheresterified forms. As used herein, the terms “ester” and “esterified”refer to the replacement of the hydrogen in the carboxylic acid group ofa PUFA molecule with another substituent. Typical esters are known tothose in the art, a discussion of which is provided by Higuchi, T. etal., Pro-drugs as Novel Delivery Systems, Vol. 14, A.C.S. SymposiumSeries, Bioreversible Carriers in Drug Design, Edward B. Roche ed.,Amer. Pharma. Assoc., Pergamon Press (1987), and Protective Groups inOrganic Chemistry, McOmie ed., Plenum Press, New York (1973), each ofwhich is incorporated herein by reference in its entirety. Examples ofcommon esters include methyl, ethyl, trichloroethyl, propyl, butyl,pentyl, tert-butyl, benzyl, nitrobenzyl, methoxybenzyl and benzhydryl.

In some embodiments, a lipid comprises at least 10%, at least 20%, atleast 30%, at least 35%, at least 40%, at least 50%, at least 60%, atleast 70% or at least 80% by weight PUFA. In some embodiments, a lipidcomprises at least 10%, at least 20%, at least 30%, at least 35%, atleast 40%, at least 50%, at least 60%, at least 70% or at least 80% byweight DHA. In some embodiments, a lipid comprises less than 50%, lessthan 40%, less than 30%, less than 20%, less than 15%, less than 10%, orless than 5% by weight EPA. In some embodiments, a lipid comprises lessthan 10%, less than 5%, less than 2%, less than 1%, or less than 0.5% byweight sterols. In some embodiments, one or more PUFAs are present in alipid in one or more forms, such as triglycerides, diglycerides,monoglycerides, phospholipids, free fatty acids, esterified fatty acids,alkali metal salts of fatty acids, alkali earth metal salts of fattyacids, and combinations thereof.

In some embodiments, a lipid separated after centrifuging in a processof the present invention comprises at least 50%, at least 60%, at least70%, at least 80%, at least 90%, at least 95%, or 50% to 95%, 50% to90%, 50% to 85%, 50% to 80%, 50% to 75%, 60% to 95%, 60% to 90%, 60% to85%, 70% to 95%, 70% to 90%, 70% to 85%, 75% to 95%, 75% to 90%, or 75%to 85%, by weight of triglycerides.

In some embodiments, the triglycerides comprise at least 10%, at least20%, at least 30%, at least 35%, at least 40%, at least 50%, at least60%, at least 70% or at least 80% by weight DHA. In some embodiments,the triglycerides comprise at least 50%, at least 40%, at least 30%, atleast 20%, at least 15%, at least 10%, or at least 5% by weight EPA.

As discussed herein, additional refining of a lipid after thecentrifuging can provide a lipid comprising at least 80%, at least 85%,at least 90%, at least 95%, at least 99%, or 80% to 99.5%, 80% to 99%,80% to 97%, 80% to 95%, 80% to 90%, 85% to 99.5%, 85% to 99%, 85% to97%, 85% to 95%, 85% to 90%, 90% to 99.5%, 90% to 99%, 90% to 97%, 90%to 95%, 95% to 99.5%, 95% to 99%, 95% to 97%, 97% to 99.5%, or 98% to99.5% triglyceride by weight.

As used herein, a “cell” refers to a lipid-containing biomaterial, suchas biomaterial derived from plants or microorganisms. In someembodiments, suitable plant material includes, but is not limited to,plant parts and oilseeds. Oilseeds include, but are not limited to,sunflower seeds, canola seeds, rapeseeds, linseeds, castor oil seeds,coriander seeds, calendula seeds or the like, and genetically modifiedvariants thereof. Oil produced from plant material and/ormicroorganisms, such as oleaginous microorganisms, according to theprocesses described herein, is also referred to as vegetable oil. Oilproduced from algae and/or fungi is also referred to as algal and/orfungal oil, respectively.

As used herein, a “microbial cell” or “microorganism” refers toorganisms such as algae, bacteria, fungi, protist, and combinationsthereof, e.g., unicellular organisms. In some embodiments, a microbialcell is a eukaryotic cell. A microbial cell suitable for use with thepresent invention includes, but is not limited to, golden algae (e.g.,microorganisms of the kingdom Stramenopiles), green algae, diatoms,dinoflagellates (e.g., microorganisms of the order Dinophyceae includingmembers of the genus Crypthecodinium such as, for example,Crypthecodinium cohnii or C. cohnii), yeast (Ascomycetes orBasidiomycetes), and fungi of the genera Mucor and Mortierella,including but not limited to Mortierella alpina and Mortierella sect.schmuckeri. A microbial cell suitable for use with the present inventioncan further include, but is not limited to genera found in the followinggroups of organisms: Stramenopiles, Hamatores, Proteromonads, Opalines,Develpayella, Diplophrys, Labrinthulids, Thraustochytrids, Biosecids,Oomycetes, Hypochytridiomycetes, Commation, Reticulosphaera,Pelagomonas, Pelagococcus, Ollicola, Aureococcus, Parmales, Diatoms,Xanthophytes, Phaeophytes, Eustigmatophytes, Raphidophytes, Synurids,Axodines (including Rhizochromulinaales, Pedinellales, Dictyochales),Chrysomeridales, Sarcinochrysidales, Hydrurales, Hibberdiales, andChromulinales.

In some embodiments, a microbial cell for use with the present inventionis a microorganism of the phylum Labyrinthulomycota. In someembodiments, a microbial cell of the phylum Labyrinthulomycota is athraustochytrid, such as a Schizochytrium or Thraustochytrium. Accordingto the present invention, the term “thraustochytrid” refers to anymember of the order Thraustochytriales, which includes the familyThraustochytriaceae, and the term “labyrinthulid” refers to any memberof the order Labyrinthulales, which includes the familyLabyrinthulaceae.

Members of the family Labyrinthulaceae were previously considered to bemembers of the order Thraustochytriales, but in more recent revisions ofthe taxonomic classification of such organisms, the familyLabyrinthulaceae is now considered to be a member of the orderLabyrinthulales. Both Labyrinthulales and Thraustochytriales areconsidered to be members of the phylum Labyrinthulomycota. Taxonomictheorists now generally place both of these groups of microorganismswith the algae or algae-like protists of the Stramenopile lineage. Thecurrent taxonomic placement of the thraustochytrids and labyrinthulidscan be summarized as follows:

Realm: Stramenopila (Chromista)

-   -   Phylum: Labyrinthulomycota (Heterokonta)        -   Class: Labyrinthulomycetes (Labyrinthulae)            -   Order: Labyrinthulales                -   Family: Labyrinthulaceae            -   Order: Thraustochytriales                -   Family: Thraustochytriaceae

For purposes of the present invention, strains of microbial cellsdescribed as thraustochytrids include the following organisms: Order:Thraustochytriales; Family: Thraustochytriaceae; Genera:Thraustochytrium (Species: sp., arudimentale, aureum, benthicola,globosum, kinnei, motivum, multirudimentale, pachydermum, proliferum,roseum, and striatum), Ulkenia (Species: sp., amoeboidea, kerguelensis,minuta, profunda, radiata, sailens, sarkariana, schizochytrops,visurgensis, yorkensis, and sp. BP-5601), Schizochytrium (Species: sp.,aggregatum, limnaceum, mangrovei, minutum, and octosporum),Japonochytrium (Species: sp., marinum), Aplanochytrium (Species: sp.,haliotidis, kerguelensis, profunda, and stocchinoi), Althornia (Species:sp., crouchii), or Elina (Species: sp., marisalba, and sinorifica). Forthe purposes of this invention, species described within Ulkenia will beconsidered to be members of the genus Thraustochytrium.Aurantiacochytrium and Oblogospora are two additional genusesencompassed by the phylum Labyrinthulomycota in the present invention.In some embodiments, a microbial cell is of the genus Thraustochystrium,Schizochytrium, and mixtures thereof.

Microbial cells suitable for use with the present invention include, butare not limited to, Labyrinthulids selected from: Order:Labyrinthulales, Family: Labyrinthulaceae, Genera: Labyrinthula(Species: sp., algeriensis, coenocystis, chattonii, macrocystis,macrocystis atlantica, macrocystis macrocystis, marina, minuta,roscoffensis, valkanovii, vitellina, vitellina pacifica, vitellinavellina, and zopfii), Labyrinthuloides (Species: sp., haliotidis, andyorkensis), Labyrinthomyxa (Species: sp., marina), Diplophrys (Species:sp., archeri), Pyrrhosorus (Species: sp., marinus), Sorodiplophrys(Species: sp., stercorea), and Chlamydomyxa (Species: sp.,labyrinthuloides, and montana) (although there is currently not aconsensus on the exact taxonomic placement of Pyrrhosorus,Sorodiplophrys, and Chlamydomyxa).

Host cells of the phylum Labyrinthulomycota include, but are not limitedto, deposited strains PTA-10212, PTA-10213, PTA-10214, PTA-10215,PTA-9695, PTA-9696, PTA-9697, PTA-9698, PTA-10208, PTA-10209, PTA-10210,PTA-10211, the microorganism deposited as SAM2179 (named “UlkeniaSAM2179” by the depositor), any Thraustochytrium species (includingformer Ulkenia species such as U. visurgensis, U. amoeboida, U.sarkariana, U. profunda, U. radiata, U. minuta and Ulkenia sp. BP-5601),and including Thraustochytrium striatum, Thraustochytrium aureum,Thraustochytrium roseum; and any Japonochytrium species. Strains ofThraustochytriales include, but are not limited to Thraustochytrium sp.(23B) (ATCC 20891); Thraustochytrium striatum (Schneider)(ATCC 24473);Thraustochytrium aureum (Goldstein) (ATCC 34304); Thraustochytriumroseum (Goldstein) (ATCC 28210); Japonochytrium sp. (L1) (ATCC 28207);ATCC 20890; ATCC 20892; a mutant strain derived from any of theaforementioned microorganisms; and mixtures thereof. Schizochytriuminclude, but are not limited to Schizochytrium aggregatum,Schizochytrium limacinum, Schizochytrium sp. (S31) (ATCC 20888),Schizochytrium sp. (S8) (ATCC 20889), Schizochytrium sp. (LC-RM) (ATCC18915), Schizochytrium sp, (SR 21), deposited strain ATCC 28209,deposited Schizochytrium limacinum strain IFO 32693, a mutant strainderived from any of the aforementioned microorganisms, and mixturesthereof. In some embodiments, the host cell is a Schizochytrium or aThraustochytrium. Schizochytrium can replicate both by successivebipartition and by forming sporangia, which ultimately releasezoospores. Thraustochytrium, however, replicate only by formingsporangia, which then release zoospores. In some embodiments, the hostcell of the invention is a recombinant host cell.

Effective culture conditions for a microbial cell for use with theinvention include, but are not limited to, effective media, bioreactor,temperature, pH, and oxygen conditions that permit lipid production. Aneffective medium refers to any medium in which a microbial cell, e.g.,Thraustochytriales microbial cell, is typically cultured. Such mediatypically comprises an aqueous medium having assimilable carbon,nitrogen, and phosphate sources, as well as appropriate salts, minerals,metals, and other nutrients, such as vitamins. Microbial cells for usewith the present invention can be cultured in conventional fermentationbioreactors, shake flasks, test tubes, microtiter dishes, and petriplates. In some embodiments, culturing is carried out at a temperature,pH, and oxygen content appropriate for a recombinant cell.

In some embodiments, a microbial cell is capable of growth at a salinitylevel of 12 g/L or less, 5 g/L or less, or 3 g/L or less of sodiumchloride.

In some embodiments, a microbial cell produces a lipid comprisingomega-3 and/or omega-6 PUFAs. In some embodiments, a microbial cellproduces a lipid comprising DHA, DPA (n-3), DPA (n-6), EPA, arachidonicacid (ARA), or the like, and combinations thereof. Non-limiting examplesof microorganisms that produce a lipid comprising a PUFA are disclosedabove and are also found in U.S. Pat. Nos. 5,340,594, 5,340,742 and5,583,019, each of which is incorporated by reference herein in itsentirety.

In some embodiments, a microbial cell comprises at least 30% by weightlipids, at least 35% by weight lipids, at least 40% by weight lipids, atleast 50% by weight lipids, at least 60% by weight lipids, at least 70%by weight lipids, or at least 80% by weight lipids. In some embodiments,a microbial cell for use with the present invention is capable ofproducing at least 0.1 grams per liter per hour (g/L/h) of DHA, at least0.2 g/L/h of DHA, at least 0.3 g/L/h of DHA, or at least 0.4 g/L/h ofDHA.

Processes

The processes of the present invention comprises lysing a cell or cellbiomass to form a lysed cell composition. As used herein, the term “cellbiomass” refers to a population of plant or microbial cells. As usedherein, the terms “lyse” and “lysing” refer to a process of rupturingthe cell wall and/or cell membrane of a cell. In some embodiments,lysing comprises a process such as: mechanically treating, chemicallytreating, enzymatically treating, physically treating, or combinationsthereof.

As used herein, mechanically treating includes, but is not limited to,homogenizing a cell, applying ultrasound to a cell, cold-pressing acell, milling a cell or the like, and combinations thereof. In someembodiments, a process comprises lysing the cell by homogenization. Insome embodiments, a process comprises lysing the cell with ahomogenizer.

Homogenizing a cell can include, but is not limited to, processesutilizing a French pressure cell press, a sonicator, a homogenizer, aball mill, a rod mill, a pebble mill, a bead mill, a high pressuregrinding roll, a vertical shaft impactor, an industrial blender, a highshear mixer, a paddle mixer, a polytron homogenizer or the like, andcombinations thereof. In some embodiments, a cell is flowed through ahomogenizer that is optionally heated. In some embodiments, suitablehomogenization can include 1 to 3 passes through a homogenizer at eitherhigh and/or low pressures. In some embodiments, a pressure duringhomogenization can be 150 bar to 1,400 bar, 150 bar to 1,200 bar, 150bar to 900 bar, 150 bar to 300 bar, 300 bar to 1,400 bar, 300 bar to1,200 bar, 300 bar to 900 bar, 400 bar to 800 bar, 500 bar to 700 bar,or 600 bar.

As used herein, physically treating can include, but is not limited to,heating a cell, drying a cell, or the like, and combinations thereof.

Heating a cell can include, but is not limited to, resistive heating,convection heating, steam heating, heating in a fluid bath, heating withsolar energy, heating with focused solar energy, and the like, any ofwhich can be performed in a tank, pool, tube, conduit, flask, or othercontainment device. In some embodiments, a cell is heated in a tank thatincludes resistive coils in/on its walls. In some embodiments, a cell isheated in a liquid bath that includes a tubing passing there through.

Drying a cell can include, but is not limited to, exposing to air flow,exposing to heat (e.g., convection heat, a heated surface, and thelike), exposing to solar energy, freeze drying (lyophilizing), spraydrying, and combinations thereof. In some embodiments, drying comprisesapplying a cell to a rotating drum that is optionally heated.

As used herein, chemically treating includes, but is not limited to,raising a pH of a cell, contacting a cell with a chemical or the like.

Raising a pH of a cell can include, but is not limited to, adding a baseto a cell composition. In some embodiments, bases suitable for use withthe present invention include, but are not limited to, hydroxide bases(e.g., LiOH, NaOH, KOH, Ca(OH)₂, and the like, and combinationsthereof), carbonate bases (e.g., Na₂CO₃, K₂CO₃, MgCO₃, and the like, andcombinations thereof), bicarbonate bases (e.g., LiHCO₃, NaHCO₃, KHCO₃,and the like, and combinations thereof), and combinations thereof. Abase can be in the form of a solid (e.g., crystals, a granulate,pellets, and the like) or a liquid (e.g., an aqueous solution, analcoholic solution such as a hydroxide base in methanol, ethanol,propanol, and the like), and combinations thereof. In some embodiments,the pH of the cell composition is raised to 8 or above, 9 or above, 10or above, 11 or above, 12 or above, or a pH of 7 to 13, 7 to 12, 7 to11, 7 to 10, 7 to 9, 8 to 13, 8 to 12, 8 to 11, 8 to 10, 8 to 9, 9 to12, 9 to 11, 9 to 10, 10 to 12, or 10 to 11.

In some embodiments, raising a pH of a cell can include, but is notlimited to, performing a chloralkali process. In some embodiments, afermentation broth containing sodium chloride and a cell composition issubjected to electrolysis, which would result in the formation of sodiumhydroxide. The formation of sodium hydroxide raises the pH of the cell.In some embodiments, a fermentation broth can include calcium chlorideor potassium chloride in place of or in addition to sodium chloride.Subjecting such a fermentation broth to electrolysis results in theformation of calcium hydroxide or potassium hydroxide, respectively,thereby raising the pH of the cell.

Enzymatic lysing refers to lysis of a cell wall or cell membrane of acell by contacting the cell with one or more enzymes. Enzymes suitablefor use with the present invention include, but are not limited to,proteases, cellulases, hemicellulases, chitinases, pectinases, andcombinations thereof. Non-limiting examples of proteases include serineproteases, theronine proteases, cysteine proteases, aspartate proteases,metalloproteases, glutamic acid proteases, alacase, and combinationsthereof. Non-limiting examples of cellulases include sucrase, maltase,lactase, alpha-glucosidase, beta-glucosidase, amylase, lysozyme,neuraminidase, galactosidase, alpha-mannosidase, glucuronidase,hyaluronidase, pullulanase, glucocerebrosidase, galactosylceramidase,acetylgalactosaminidase, facosidase, hexosaminidase, iduronidase,maltase-glucoamylase, and combinations thereof. A non-limiting exampleof a chitinase includes chitotriosidase. Non-limiting examples ofpectinases include pectolyase, pectozyme, polygalacturonase, andcombinations thereof. In some embodiments, some enzymes are activated byheating.

As used herein, a “lysed cell composition” refers to a compositioncomprising one or more lysed cells, including cell debris and othercontents of the cell, in combination with a lipid (from the lysedcells), and optionally, broth that contains microbial cells or plantmaterial. In some embodiments, plant material is contained in a broth ormedia comprising the plant material and water. In some embodiments, amicrobial cell is contained in a fermentation broth or media comprisingthe microbial cell and water. In some embodiments, a lysed cellcomposition refers to a composition comprising one or more lysed cells,cell debris, a lipid, the natural contents of the cell, and aqueouscomponents from a broth. In some embodiments, a lysed cell compositionis in the form of an oil-in-water emulsion comprising a mixture of acontinuous aqueous phase and a dispersed lipid phase. In someembodiments, a dispersed lipid phase is present in a concentration of 1%to 60%, 1% to 50%, 1% to 40%, 1% to 30%, 1% to 20%, 5% to 60%, 5% to50%, 5% to 40%, 5% to 30%, 5% to 20%, 10% to 60%, 10% to 50%, 10% to40%, 20% to 60%, 20% to 50%, 20% to 40%, 30% to 60%, 30% to 50%, or 40%to 60% by weight of an emulsified lysed cell composition.

While not being bound to any particular theory, it is believed theprocesses of the present invention break up or demulsify an emulsifiedlysed cell composition, allowing a lipid to be separated from the lysedcell composition. As used herein, the terms “emulsion” and “emulsified”refers to a mixture of two or more immiscible phases or layers whereinone phase or layer is dispersed in another phase or layer. As usedherein, the terms “break,” “break up,” “demulsify,” “demulsification,”“demulsifying,” and “breaking” refer to a process of separatingimmiscible phases or layers of an emulsion. For example, demulsifying orbreaking an emulsified lysed cell composition refers to a process bywhich an emulsified lysed cell composition changes from an emulsionhaving one or more phases or layers to a composition having two or morephases or layers. For example, in some embodiments, a process of thepresent invention breaks an emulsified lysed cell composition from asingle-phase to two or more phases. In some embodiments, the two or morephases include a lipid phase and an aqueous phase. In some embodiments,a process of the present invention breaks an emulsified lysed cellcompositions from one or more phases to at least three phases. In someembodiments, the three phases include a lipid phase, an aqueous phase,and a solid phase. In some embodiments, the three phases include a lipidphase, an emulsion phase, and an aqueous phase.

In some embodiments, the processes of the present invention demulsify alysed cell composition to form a demulsified cell composition byremoving or breaking at least 75% of the emulsion, at least 80% of theemulsion, at least 85% of the emulsion, at least 90% of the emulsion, atleast 95% of the emulsion, at least 99% of the emulsion. In someembodiments, the process of the present invention demulsify a lysed cellcomposition by removing or breaking 75% of the emulsion to 99% of theemulsion, 75% of the emulsion to 95% of the emulsion, 75% of theemulsion to 90% of the emulsion, 75% of the emulsion to 85% of theemulsion, 75% of the emulsion to 80% of the emulsion, 80% of theemulsion to 99% of the emulsion, 80% of the emulsion to 95% of theemulsion, 80% of the emulsion to 90% of the emulsion, 80% of theemulsion to 85% of the emulsion, 85% of the emulsion to 99% of theemulsion, 85% of the emulsion to 95% of the emulsion, 85% of theemulsion to 90% of the emulsion, 90% of the emulsion to 99% of theemulsion, 90% of the emulsion to 95% of the emulsion, or 95% of theemulsion to 99% of the emulsion by weight or volume.

In some embodiments, prior to lysing the cell, the cell can be washedand/or pasteurized. In some embodiments, washing the cell includes usingan aqueous solution, such as water, to remove any extracellularwater-soluble or water-dispersible compounds. In some embodiments, thecell can be washed once, twice, thrice, or more. In some embodiments,pasteurizing the cell includes heating the cell to inactivate anyundesirable enzymes, for example any enzymes that might degrade lipid orreduce the yield of PUFAs. In some embodiments, the cell can be washedfirst and then pasteurized.

In some embodiments, the cell is plant biomaterial and the plantbiomaterial is formed prior to lysing. In some embodiments, the plantbiomaterial is formed by removing or extracting oilseeds from a plant.In some embodiments, an interior of an oilseed is removed from an outerhull of an oilseed by grinding, milling, extruding, aspirating,crushing, or combinations thereof. In some embodiments, the dehulledoilseeds can be homogenized or expelled using processes known in theart, such as by passing the oilseeds through a press to grind thedehulled oilseeds into a cake. In some embodiments, water can be addedto the cake to form an emulsified lysed cell composition. In someembodiments, the emulsified lysed cell composition can be filtered usingprocesses known in the art to remove any excess hull fragments from thelysed cell composition.

In some embodiments, treating a lysed cell composition with a first basebreaks up (i.e., demulsifies) an emulsified lysed cell composition. Insome embodiments, treating a lysed cell composition with a second basebreaks (i.e., demulsifies) an emulsified lysed cell composition. In someembodiments, treating a lysed cell composition with a salt breaks (i.e.,demulsifies) an emulsified lysed cell composition. In some embodiments,heating a lysed cell composition breaks (i.e., demulsifies) anemulsified lysed cell composition. In some embodiments, agitating alysed cell composition breaks (i.e., demulsifies) an emulsified lysedcell composition. In some embodiments, simultaneous heating andagitating of a lysed cell composition breaks (i.e., demulsifies) anemulsified lysed cell composition. In some embodiments, one or more ofthe preceding treatments breaks up (i.e., demulsifies) an emulsifiedlysed cell composition.

In some embodiments, the process of the invention comprises raising thepH of a cell composition to lyse and/or demulsify the cell composition.In some embodiments, the process of the invention comprises raising thepH of a lysed cell composition to demulsify the lysed cell composition.In some embodiments, raising the pH comprises contacting a cellcomposition or lysed cell composition with a base. In some embodiments,the process of the invention comprises contacting a lysed cellcomposition with a base to demulsify the lysed cell composition. As usedherein, “contacting” refers to combining a cell composition or a lysedcell composition with a second composition (e.g., by adding acomposition to a cell composition or a lysed cell composition, by addinga cell composition or a lysed cell composition to a composition, and thelike). As used herein, a “composition” can comprise a pure material orinclude a combination of two or more materials, substances, excipients,portions, and the like. Contacting a lysed cell composition with a firstbase raises the pH of the lysed cell composition. In some embodiments, alysed cell composition is contacted with a second base. In someembodiments, the pH of a lysed cell composition or a demulsified cellcomposition is raised a second time. In some embodiments, the secondraising of the pH comprises contacting a lysed cell composition ordemulsified cell composition with a second base. In some embodiments, alysed cell composition is contacted with a first base, then heated,agitated, or a combination thereof, and subsequently contacted with asecond base to provide a treated lysed cell emulsion.

In some embodiments, the first base and/or second base has a pK_(b) of 1to 12, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 2 to 12, 2 to 10, 2 to 8, 2 to6, 2 to 5, 3 to 10, 3 to 6, 3 to 5, 4 to 10, 4 to 8, 4 to 6, 5 to 10, or5 to 8. As used herein, the term “pK_(b)” refers to the negativelogarithm of the base association constant, K_(b), of the base. K_(b)refers to the equilibrium constant for the ionization of the base inwater, wherein:

B+H₂O

HB⁺+OH⁻; and

the K_(b) of base, B, is defined as:

$K_{b} = {\frac{\left\lbrack {HB}^{+} \right\rbrack \left\lbrack {OH}^{-} \right\rbrack}{\lbrack B\rbrack}.}$

Bases suitable for use with the present invention include, but are notlimited to, hydroxide bases (e.g., LiOH, NaOH, KOH, Ca(OH)₂, and thelike, and combinations thereof), carbonate bases (e.g., Na₂CO₃, K₂CO₃,MgCO₃, and the like, and combinations thereof), bicarbonate bases (e.g.,LiHCO₃, NaHCO₃, KHCO₃, and the like, and combinations thereof), andcombinations thereof. A base can be in the form of a solid (e.g.,crystals, a granulate, pellets, and the like) or a liquid (e.g., anaqueous solution, an alcoholic solution such as a hydroxide base inmethanol, ethanol, propanol, and the like), and combinations thereof.Thus, a solvent can be optionally present in a base for use with thepresent invention. As used herein, “solvent” refers to an agent that ishydrophobic or lipophilic. As used herein, “hydrophobic” refers to anagent that is repelled from a mass of water. As used herein,“lipophilic” refers to an agent that dissolves in lipids.

In some embodiments, contacting a cell composition or a lysed cellcomposition with a base raises the pH of the lysed cell composition. Insome embodiments, contacting a lysed cell composition with a base raisesthe pH of the lysed cell composition to 8 or above, 9 or above, 10 orabove, 11 or above, 12 or above, or a pH of 7 to 13, 7 to 12, 7 to 11, 7to 10, 7 to 9, 8 to 13, 8 to 12, 8 to 11, 8 to 10, 8 to 9, 9 to 12, 9 to11, 9 to 10, 10 to 12, or 10 to 11. In some embodiments, contacting alysed cell composition with a base provides a pH of 8 or below, 7 orbelow, 6 or below, or 5 or below to the composition.

In some embodiments, raising the pH of the cell composition or lysedcell composition with the addition of the base inhibits lipid oxidation,thereby minimizing the amount of free radicals in the lysed cellcomposition so that the crude lipid obtained from the processes of theinvention has a low peroxide value (e.g., 5 or less, 4.5 or less, 4 orless, 3.5 or less, 3 or less, 2.5 or less, 2 or less, 1.5 or less, 1 orless, 0.5 or less, 0.2 or less, or 0.1 or less) and/or a low anisidinevalue (e.g., 26 or less, 25 or less, 20 or less, 15 or less, 10 or less,5 or less, 2 or less, or 1 or less). As used herein, the terms “peroxidevalue” or “PV” refer to the measure of primary reaction products, suchas peroxides and hydroperoxides, that occur during oxidation of thelipid. As used herein peroxide value is measured in meq/kg. As usedherein, the terms “anisidine value” or “AV” refer to the measure ofsecondary reaction products, such as aldehydes and ketones, that occurduring oxidation of the lipid.

In some embodiments, free radicals in the lysed cell composition afteradjusting the pH with a base are detected using an Electron ParamagneticResonance spectrometer, e.g., Bruker BioSpin e-scan EPR (system numberSC0274) (Bruker BioSpin, Billerica, Mass.). In some embodiments, asample of the lysed cell composition is diluted in about 1:1 ratio withdeionized water prior to measuring the EPR. In some embodiments, inorder to measure the EPR, a spin trap chemical is added to a sample ofthe lysed cell composition. In some embodiments, the spin trap chemicalis any spin trap chemical known in the art, including, but not limitedto, POBN (α-(4-Pyridyl 1-oxide)-N-tert-butylnitrone) or DMPO(5,5-dimethyl-1-pyrroline-N-oxide). In some embodiments, the spin trapchemical is about 1.25 M and about 50 μL is added to about 0.5 gramsample of the lysed cell composition. In some embodiments, a samplecontaining the spin trap chemical is incubated at room temperature(e.g., about 20° C.). In some embodiments, the following spectrometerparameters are used: modulation frequency of about 86 Hz, modulationamplitude of about 2 gauss, microwave power of about 5 mW, time constantof about 20 seconds, sweep time of about 10 seconds, sweep width ofabout 100 gauss, and a number of scans of about 8. The EPR is measuredover time to determine the concentration of free radicals present in thelipid. In some embodiments, the EPR is measured hourly over a course offour hours. In some embodiments, the lysed cell composition has an EPRsignal strength (intensity or amplitude) at the above listed parametersof less than 0.15×10⁶, less than 0.14×10⁶, less than 0.13×10⁶, less than0.12×10⁶, less than 0.11×10⁶, less than 0.1×10⁶, less than 0.09×10⁶,less than 0.08×10⁶, less than 0.07×10⁶, less than 0.06×10⁶, or less than0.05×10⁶ after 4 hours. In some embodiments, the lysed cell compositionhas an EPR of 0.05×10⁶ to 0.15×10⁶, 0.05×10⁶ to 0.14×10⁶, 0.05×10⁶ to0.13×10⁶, 0.05×10⁶ to 0.12×10⁶, 0.05×10⁶ to 0.11×10⁶, 0.05×10⁶ to0.1×10⁶, 0.05×10⁶ to 0.09×10⁶, 0.07×10⁶ to 0.15×10⁶, 0.07×10⁶ to0.13×10⁶, 0.07×10⁶ to 0.11×10⁶, 0.08×10⁶ to 0.14×10⁶, 0.08×10⁶ to0.12×10⁶, 0.08×10⁶ to 0.1×10⁶, 0.09×10⁶ to 0.13×10⁶, or 0.09×10⁶ to0.11×10⁶. In some embodiments, the pH of the lysed cell compositionresulting in an EPR specified above is 8 to 12, 8 to 11, 8 to 10, 8 to9, 9 to 12, 9 to 11, 9 to 10, 10 to 12, or 10 to 11. In someembodiments, a lysed cell composition having an EPR signal strengthspecified above results in a crude lipid having an AV of 26 or less, 25or less, 20 or less, 15 or less, 10 or less, 5 or less, 2 or less, or 1or less. In some embodiments, a lysed cell composition having an EPRspecified above results in a crude lipid having a PV of 5 or less, 4.5or less, 4 or less, 3.5 or less, 3 or less, 2.5 or less, 2 or less, 1.5or less, 1 or less, 0.5 or less, 0.2 or less, or 0.1 or less.

In some embodiments, a process comprises contacting a cell compositionor lysed cell composition with a salt to demulsify the lysed cellcomposition. As used herein, a “salt” refers to an ionic compound formedby replacing a hydrogen ion from an acid with a metal (e.g., an alkalimetal, an alkali earth metal, a transition metal, and the like) or apositively charged compound (e.g., NH₄ ⁺ and the like). Salts suitablefor use with the present invention include, but are not limited to,alkali metal salts, alkali earth metal salts, or the like, andcombinations thereof. Negatively charged ionic species present in a saltfor use with the present include, but are not limited to, halides,sulfate, bisulfate, sulfite, phosphate, hydrogen phosphate, dihydrogenphosphate, carbonate, bicarbonate, or the like, and combinationsthereof. In some embodiments, a salt for use with the present inventionis selected from: sodium chloride, sodium sulfate, sodium carbonate,calcium chloride, potassium sulfate, magnesium sulfate, monosodiumglutamate, ammonium sulfate, potassium chloride, iron chloride, ironsulfate, aluminum sulfate, and combinations thereof. In someembodiments, a salt does not include NaOH. A salt can be added as asolid (e.g., in crystalline, amorphous, pelletized, and/or granulatedform), and/or as a solution (e.g., a dilute solution, a saturatedsolution, or a super-saturated solution) containing, for example, water,an alcohol, and the like, and combinations thereof.

In some embodiments, the salt is added in an amount of 5 g/l to 25 g/l,5 g/l to 10 g/l, 10 g/l to 15 g/l, 15 g/l to 20 g/l, 20 g/l to 25 g/l,or 10 g/l to 20 g/l.

In some embodiments, a temperature of a cell composition or a lysed cellcomposition is less than or equal to 60° C., less than or equal to 55°C., less than or equal to 45° C., less than or equal to 40° C., lessthan or equal to 35° C., less than or equal to 30° C., or less than orequal to 25° C. when a salt is added to demulsify the cell compositionor the lysed cell composition. In some embodiments, a temperature of alysed cell composition is 0° C. to 60° C., 0° C. to 55° C., 0° C. to 50°C., 0° C. to 45° C., 0° C. to 4° C., 0° C. to 35° C., 0° C. to 30° C.,0° C. to 25° C., 20° C. to 60° C., 20° C. to 55° C., 20° C. to 50° C.,20° C. to 45° C., 20° C. to 40° C., 20° C. to 35° C., 20° C. to 30° C.,30° C. to 60° C., 30° C. to 55° C., 30° C. to 50° C., 30° C. to 45° C.,30° C. to 40° C., 30° C. to 40° C., 40° C. to 60° C., 40° C. to 55° C.,40° C. to 50° C., or 50° C. to 60° C. when a salt is added to demulsifythe cell composition or the lysed cell composition.

In some embodiments, the process comprises contacting a cell compositionor a lysed cell composition with 20% or less, 15% or less, 10% or less,7.5% or less, 5% or less, or 2% or less salt by weight, of the lysedcell composition or the cell composition. In some embodiments, a processcomprises contacting a cell composition or a lysed cell composition with0.1% to 20%, 0.1% to 15%, 0.1% to 10%, 0.5% to 20%, 0.5% to 15%, 0.5% to10%, 0.5% to 5%, 0.5% to 4%, 0.5% to 3%, 0.5% to 2.5%, 0.5% to 2%, 0.5%to 1.5%, 0.5% to 1%, 1% to 20%, 1% to 15%, 1% to 10%, 1% to 5%, 1% to4%, 1% to 3%, 1% to 2.5%, 1% to 2%, 1% to 1.5%, 1.5% to 5%, 1.5% to 4%,1.5% to 3%, 1.5% to 2.5%, 1.5% to 2%, 2% to 20%, 2% to 15%, 2% to 10%,2% to 5%, 2% to 4%, 2% to 3%, 2% to 2.5%, 2.5% to 5%, 2.5% to 4%, 2.5%to 3%, 3% to 5%, 3% to 4%, 4% to 5%, 5% to 20%, 5% to 15%, 5% to 10%,10% to 20%, 10% to 15%, or 15% to 20% salt, by weight, of the cellcomposition or lysed cell composition (e.g., a total broth weight). Forexample, when a lysed cell composition weighs 1,000 kg, contacting with0.5% to 20% salt, by weight, requires combining 5 kg to 200 kg of saltwith the lysed cell composition.

In some embodiments, the process comprises heating a cell composition ora lysed cell composition to demulsify the lysed cell composition. Insome embodiments the cell composition or the lysed cell composition isheated for a sufficient period of time for a base and/or a salt todemulsify a cell composition or a lysed cell composition. In someembodiments, the process comprises heating a cell composition or a lysedcell composition for at least 5 minutes, at least 10 minutes, at least20 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, atleast 4 hours, at least 8 hours, at least 12 hours, at least 18 hours,at least 24 hours, at least 30 hours, at least 36 hours, at least 42hours, at least 48 hours, at least 54 hours, at least 60 hours, at least66 hours, at least 72 hours, at least 78 hours, at least 84 hours, atleast 90 hours or at least 96 hours. In some embodiments, the processcomprises heating a lysed cell composition for 5 minutes to 96 hours, 5minutes to 4 hours, 5 minutes to 2 hours, 5 minutes to 1 hour, 10minutes to 4 hours, 10 minutes to 2 hours, 10 minutes to 1 hour, 1 hourto 96 hours, 1 hour to 84 hours, 1 hour to 72 hours, 1 hour to 60 hours,1 hour to 48 hours, 1 hour to 36 hours, 1 hour to 24 hours, 1 hour to 4hours, 4 hours to 96 hours, 4 hours to 84 hours, 4 hours to 72 hours, 4hours to 60 hours, 4 hours to 48 hours, 4 hours to 36 hours, 4 hours to24 hours, 8 hours to 96 hours, 8 hours to 84 hours, 8 hours to 72 hours,8 hours to 60 hours, 8 hours to 48 hours, 8 hours to 36 hours, 8 hoursto 24 hours, 8 hours to 12 hours, 12 hours to 96 hours, 12 hours to 84hours, 12 hours to 72 hours, 12 hours to 60 hours, 12 hours to 48 hours,12 hours to 36 hours, 12 hours to 24 hours, 24 hours to 96 hours, 24hours to 84 hours, 24 hours to 72 hours, 24 hours to 60 hours, 24 hoursto 48 hours, or 24 hours to 36 hours.

In some embodiments, a cell composition or a lysed cell composition canbe heated at a temperature of at least 10° C., at least 20° C., at least25° C., at least 30° C., at least 35° C., at least 40° C., at least 45°C., at least 50° C., at least 55° C., at least 60° C., at least 65° C.,at least 70° C., at least 75° C., at least 80° C., at least 85° C., atleast 90° C., at least 95° C., or at least 100° C. In some embodiments,a process comprises heating a cell composition or a lysed cellcomposition at a temperature of 10° C. to 100° C., 10° C. to 90° C., 10°C. to 80° C., 10° C. to 70° C., 20° C. to 100° C., 20° C. to 90° C., 20°C. to 80° C., 20° C. to 70° C., 30° C. to 100° C., 30° C. to 90° C., 30°C. to 80° C., 30° C. to 70° C., 40° C. to 100° C., 40° C. to 90° C., 40°C. to 80° C., 50° C. to 100° C., 50° C. to 90° C., 50° C. to 80° C., 50°C. to 70° C., 60° C. to 100° C., 60° C. to 90° C., 60° C. to 80° C., 70°C. to 100° C., 70° C. to 90° C., 80° C. to 100° C., 80° C. to 90° C., or90° C. to 100° C. In some embodiments, a salt can be added to the cellcomposition or the lysed cell composition during the heating.

In some embodiments, a cell composition or a lysed cell composition canbe heated in a closed system or in a system with an evaporator. In someembodiments, a cell composition or lysed cell composition can be heatedin a system with an evaporator such that a portion of the water presentin the cell composition or the lysed cell composition is removed byevaporation. In some embodiments, a process comprises heating a cellcomposition or a lysed cell composition in a system with an evaporatorto remove up to 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% byweight of water present in the cell composition or lysed cellcomposition. In some embodiments, a process comprises heating a cellcomposition or a lysed cell composition in a system with an evaporatorto remove 1% to 50%, 1% to 45%, 1% to 40%, 1% to 35%, 1% to 30%, 1% to25%, 1% to 20%, 1% to 15%, 1% to 10%, 1% to 5%, 5% to 50%, 5% to 45%, 5%to 40%, 5% to 35%, 5% to 30%, 5% to 25%, 5% to 20%, 5% to 15%, 5% to10%, 10% to 50%, 10% to 45%, 10% to 40%, 10% to 35%, 10% to 30%, 10% to25%, 10% to 20%, 10% to 15%, 15% to 50%, 15% to 45%, 15% to 40%, 15% to35%, 15% to 30%, 15% to 25%, 15% to 20%, 20% to 50%, 20% to 45%, 20% to40%, 20% to 35%, 20% to 30%, 20% to 25%, 25% to 50%, 25% to 45%, 25% to40%, 25% to 35%, 25% to 30%, 30% to 50%, 30% to 45%, 30% to 40%, 30% to35%, 35% to 50%, 35% to 45%, 35% to 40%, 40% to 50%, 40% to 45%, or 45%to 50%.

In some embodiments, the process comprises holding a cell composition ora lysed cell composition in a vessel for a predetermined time todemulsify the lysed cell composition. In some embodiments, the processcomprises holding a cell composition or a lysed cell composition in avessel for at least 5 minutes, at least 10 minutes, at least 20 minutes,at least 30 minutes, at least 1 hour, at least 2 hours, at least 4hours, at least 8 hours, at least 12 hours, at least 18 hours, at least24 hours, at least 30 hours, at least 36 hours, at least 42 hours, atleast 48 hours, at least 54 hours, at least 60 hours, at least 66 hours,at least 72 hours, at least 78 hours, at least 84 hours, at least 90hours or at least 96 hours. In some embodiments, the process comprisesholding a cell composition or a lysed cell composition for 5 minutes to96 hours, 5 minutes to 4 hours, 5 minutes to 2 hours, 5 minutes to 1hour, 10 minutes to 4 hours, 10 minutes to 2 hours, 10 minutes to 1hour, 1 hour to 96 hours, 1 hour to 84 hours, 1 hour to 72 hours, 1 hourto 60 hours, 1 hour to 48 hours, 1 hour to 36 hours, 1 hour to 24 hours,1 hour to 4 hours, 4 hours to 96 hours, 4 hours to 84 hours, 4 hours to72 hours, 4 hours to 60 hours, 4 hours to 48 hours, 4 hours to 36 hours,4 hours to 24 hours, 8 hours to 96 hours, 8 hours to 84 hours, 8 hoursto 72 hours, 8 hours to 60 hours, 8 hours to 48 hours, 8 hours to 36hours, 8 hours to 24 hours, 8 hours to 12 hours, 12 hours to 96 hours,12 hours to 84 hours, 12 hours to 72 hours, 12 hours to 60 hours, 12hours to 48 hours, 12 hours to 36 hours, 12 hours to 24 hours, 24 hoursto 96 hours, 24 hours to 84 hours, 24 hours to 72 hours, 24 hours to 60hours, 24 hours to 48 hours, or 24 hours to 36 hours.

In some embodiments, the process comprises contacting an antioxidantwith a lysed cell emulsion. Antioxidants suitable for use with thepresent invention include, but are not limited to, a tocopherol, atocotrienol, a polyphenol, resveratrol, a flavonoid, a carotenoid,lycopene, a carotene, lutein, ascorbic acid, ascorbyl palmitate, or thelike, and combinations thereof.

In some embodiments, the process comprises allowing an emulsified lysedcell composition to stand, wherein the lipid is separated from theemulsified lysed cell composition using gravity.

As used herein, the terms “agitating” and “agitation” refer to a processof affecting motion in a lysed cell composition through an applicationof force. In some embodiments, the process of the invention comprisesagitating a cell composition or a lysed cell composition by stirring,mixing, blending, shaking, vibrating, or a combination thereof. In someembodiments, the process of agitating a cell composition or a lysed cellcomposition demulsifies the cell composition or the lysed cellcomposition.

In some embodiments, the process of the invention comprises agitating alysed cell composition at 0.1 hp/1,000 gal to 10 hp/1,000 gal, 0.5hp/1,000 gal to 8 hp/1,000 gal, 1 hp/1,000 gal to 6 hp/1,000 gal, or 2hp/1,000 gal to 5 hp/1,000 gal of lysed cell composition.

In some embodiments, the process of the invention comprises agitating acell composition or a lysed cell composition using an agitator. In someembodiments, the agitator is a dispersion style agitator that dispersesa base and/or salt in the cell composition or the lysed cellcomposition. In some embodiments, an agitator has one or more impellers.As used herein, “impeller” refers to a device arranged to impart motionto a cell composition or a lysed cell composition when rotated.Impellers suitable for use with the present invention include straightblade impellers, Rushton blade impellers, axial flow impellers, radialflow impellers, concave blade disc impellers, high-efficiency impellers,propellers, paddles, turbines, or the like, and combinations thereof. Insome embodiments, a process includes agitating a cell composition or alysed cell composition using an agitator having an impeller tip speed of90 ft/min to 1,200 ft/min, 200 ft/min to 1,000 ft/min, 300 ft/min to 800ft/min, 400 ft/min to 700 ft/min, or 500 ft/min to 600 ft/min. In someembodiments, a process includes agitating a cell composition or a lysedcell composition using an agitator having an impeller tip speed of 350centimeters/second to 900 centimeters per second, 350 centimeters/secondto 850 centimeters per second, 350 centimeters/second to 800centimeters/second, 350 centimeters/second to 750 centimeters/second,350 centimeters/second to 700 centimeters/second, 350 centimeters/secondto 650 centimeters/second, 350 centimeters/second to 600centimeters/second, 350 centimeters/second to 550 centimeters/second,350 centimeters/second to 500 centimeters/second, 350 centimeters/secondto 450 centimeters/second, 350 centimeters/second to 400centimeters/second, 400 centimeters/second to 900 centimeters persecond, 400 centimeters/second to 850 centimeters per second, 400centimeters/second to 800 centimeters/second, 400 centimeters/second to750 centimeters/second, 400 centimeters/second to 700centimeters/second, 400 centimeters/second to 650 centimeters/second,400 centimeters/second to 600 centimeters/second, 400 centimeters/secondto 550 centimeters/second, 400 centimeters/second to 500centimeters/second, 400 centimeters/second to 450 centimeters/second,450 centimeters/second to 900 centimeters per second, 450centimeters/second to 850 centimeters per second, 450 centimeters/secondto 800 centimeters/second, 450 centimeters/second to 750centimeters/second, 450 centimeters/second to 700 centimeters/second,450 centimeters/second to 650 centimeters/second, 450 centimeters/secondto 600 centimeters/second, 450 centimeters/second to 550centimeters/second, 450 centimeters/second to 500 centimeters/second,500 centimeters/second to 900 centimeters per second, 500centimeters/second to 850 centimeters per second, 500 centimeters/secondto 800 centimeters/second, 500 centimeters/second to 750centimeters/second, 500 centimeters/second to 700 centimeters/second,500 centimeters/second to 650 centimeters/second, 500 centimeters/secondto 600 centimeters/second, 500 centimeters/second to 550centimeters/second, 550 centimeters/second to 900 centimeters persecond, 550 centimeters/second to 850 centimeters per second, 550centimeters/second to 800 centimeters/second, 550 centimeters/second to750 centimeters/second, 550 centimeters/second to 700centimeters/second, 550 centimeters/second to 650 centimeters/second,550 centimeters/second to 600 centimeters/second, 600 centimeters/secondto 900 centimeters per second, 600 centimeters/second to 850 centimetersper second, 600 centimeters/second to 800 centimeters/second, 600centimeters/second to 750 centimeters/second, 600 centimeters/second to700 centimeters/second, 600 centimeters/second to 650centimeters/second, 650 centimeters/second to 900 centimeters persecond, 650 centimeters/second to 850 centimeters per second, 650centimeters/second to 800 centimeters/second, 650 centimeters/second to750 centimeters/second, 650 centimeters/second to 700centimeters/second, 700 centimeters/second to 900 centimeters persecond, 700 centimeters/second to 850 centimeters per second, 700centimeters/second to 800 centimeters/second, 700 centimeters/second to750 centimeters/second, 750 centimeters/second to 900 centimeters persecond, 750 centimeters/second to 850 centimeters per second, 750centimeters/second to 800 centimeters/second, 800 centimeters/second to900 centimeters per second, 800 centimeters/second to 850 centimetersper second, or 850 centimeters/second to 900 centimeters/second. As usedherein, “impeller tip speed” refers to the speed of the outer mostportion of the impeller as it rotates around its central axis.

In some embodiments, the agitating (and optionally additional steps asdescribed herein) is performed in a container comprising an impeller,wherein a ratio of the impeller diameter to the container volume is 0.1to 0.5, 0.1 to 0.4, 0.2 to 0.5, 0.2 to 0.4, 0.3 to 0.5, or 0.3 to 0.4.

In some embodiments, the agitating (and optionally additional steps asdescribed herein) is performed in a container comprising an impeller,wherein a ratio of the impeller diameter to the inner diameter of thecontainer is at least 0.25, at least 0.34, at least 0.65, 0.25 to 0.65,0.25 to 0.33, 0.3 to 0.6, 0.3 to 0.5, 0.3 to 0.4, 0.34 to 0.65, 0.34 to0.6, 0.34 to 0.55, 0.37 to 0.55, 0.4 to 0.65, 0.4 to 0.6, 0.4 to 0.5, or0.42 to 0.55.

In some embodiments, agitating comprises mixing a cell composition or alysed cell composition such that the cell composition or the lysed cellcomposition is placed under flow conditions described by a Reynoldsnumber of 10 to 10,000, 1,000 to 10,000, 1,500 to 10,000, or 2,000 to10,000. In some embodiments, a lysed cell emulsion during the agitatinghas a Reynolds number of 2,000 or more, 3,000 or more, or 5,000 or more,or 2,000 to 10,000, 3,000 to 10,000, or 5,000 to 10,000.

In some embodiments, a process comprises agitating a cell composition ora lysed cell composition for at least 5 minutes, at least 10 minutes, atleast 20 minutes, at least 30 minutes, at least 1 hour, at least 2hours, at least 4 hours, at least 8 hours, at least 12 hours, at least18 hours, at least 24 hours, at least 30 hours, at least 36 hours, atleast 42 hours, at least 48 hours, at least 54 hours, at least 60 hours,at least 66 hours, at least 72 hours, at least 78 hours, at least 84hours, at least 90 hours or at least 96 hours. In some embodiments, aprocess comprises agitating a cell composition or a lysed cellcomposition for 5 minutes to 96 hours, 5 minutes to 4 hours, 5 minutesto 2 hours, 5 minutes to 1 hour, 10 minutes to 4 hours, 10 minutes to 2hours, 10 minutes to 1 hour, 1 hour to 96 hours, 1 hour to 84 hours, 1hour to 72 hours, 1 hour to 60 hours, 1 hour to 48 hours, 1 hour to 36hours, 1 hour to 24 hours, 1 hour to 4 hours, 4 hours to 96 hours, 4hours to 84 hours, 4 hours to 72 hours, 4 hours to 60 hours, 4 hours to48 hours, 4 hours to 36 hours, 4 hours to 24 hours, 8 hours to 96 hours,8 hours to 84 hours, 8 hours to 72 hours, 8 hours to 60 hours, 8 hoursto 48 hours, 8 hours to 36 hours, 8 hours to 24 hours, 8 hours to 12hours, 12 hours to 96 hours, 12 hours to 84 hours, 12 hours to 72 hours,12 hours to 60 hours, 12 hours to 48 hours, 12 hours to 36 hours, 12hours to 24 hours, 20 hours to 40 hours, 24 hours to 96 hours, 24 hoursto 84 hours, 24 hours to 72 hours, 24 hours to 60 hours, 24 hours to 48hours, or 24 hours to 36 hours.

In some embodiments, a process comprises simultaneously agitating andheating a cell composition or a lysed cell composition to demulsify thecell composition or the lysed cell composition. In some embodiments, aprocess comprises agitating a cell composition or a lysed cellcomposition at a temperature of at least 10° C., at least 20° C., atleast 25° C., at least 30° C., at least 35° C., at least 40° C., atleast 45° C., at least 50° C., at least 55° C., at least 60° C., atleast 65° C., at least 70° C., at least 75° C., at least 80° C., atleast 85° C., at least 90° C., at least 95° C., or at least 100° C. Insome embodiments, a process comprises agitating a cell composition or alysed cell composition at a temperature of 10° C. to 100° C., 10° C. to90° C., 10° C. to 80° C., 10° C. to 70° C., 20° C. to 100° C., 20° C. to90° C., 20° C. to 80° C., 20° C. to 70° C., 30° C. to 100° C., 30° C. to90° C., 30° C. to 80° C., 30° C. to 70° C., 40° C. to 100° C., 40° C. to90° C., 40° C. to 80° C., 50° C. to 100° C., 50° C. to 90° C., 50° C. to80° C., 50° C. to 70° C., 60° C. to 100° C., 60° C. to 90° C., 60° C. to80° C., 70° C. to 100° C., 70° C. to 90° C., 80° C. to 1000° C., 80° C.to 90° C., or 90° C. to 100° C.

In some embodiments, the various combinations of forming a lysed cellcomposition, contacting a lysed cell composition with a base or raisingthe pH of a lysed cell composition, contacting a lysed cell compositionwith a salt, heating the lysed cell composition, and agitating a lysedcell composition can occur in a single vessel. In some embodiments, thevarious combinations of forming a cell composition, contacting a cellcomposition with a base or raising the pH of a cell composition,contacting a cell composition with a salt, heating the cell composition,and agitating a cell composition can occur in a single vessel. In someembodiments, the single vessel includes a fermentation vessel. In someembodiments, the fermentation vessel can have a volume of at least20,000 liters, at least 50,000 liters, at least 100,000 liters, at least120,000 liters, at least 150,000 liters, at least 200,000 liters, or atleast 220,000 liters. In some embodiments, the fermentation vessel canhave a volume of 20,000 liters to 220,000 liters, 20,000 liters to100,000 liters, 20,000 liters to 50,000 liters, 50,000 liters to 220,000liters, 50,000 liters to 150,000 liters, 50,000 liters to 100,000liters, 100,000 liters to 220,000 liters, 100,000 liters to 150,000liters, 100,000 liters to 120,000 liters, 150,000 liters to 220,000liters, 150,000 liters to 200,000 liters, or 200,000 liters to 220,000liters.

In some embodiments, a quantity of cell composition or lysed cellcomposition formed in a vessel can be transferred into one or moreagitation vessels. In some embodiments, the agitation vessels can have avolume of at least 20,000 liters, at least 30,000 liters, at least40,000 liters or at least 50,000 liters. In some embodiments, theagitation vessels can have a volume of 20,000 liters to 50,000 liters,20,000 liters to 40,000 liters, 20,000 liters to 30,000 liters, 30,000liters to 50,000 liters, 30,000 liters to 40,000 liters or 40,000 litersto 50,000 liters.

In some embodiments, the agitation vessels can have any combination ofthe following properties. In some embodiments, the agitation vessels canhave two impellers. In some embodiments, the impellers are Rushton bladeimpellers. In some embodiments, the impellers are separated from eachother by a distance at least equal to a diameter of the smallestimpeller. In some embodiments, the impellers are 30 inches to 40 inches,33 inches to 37 inches, 33 inches, 34 inches, 35 inches, 36 inches or 37inches from tip to tip. In some embodiments, the agitation vessels havea volume of at least 10,000 liters, at least 20,000 liters, at least30,000 liters, at least 40,000 liters or at least 50,000 liters. In someembodiments, the agitation vessels have an inner diameter of 90 inchesto 110 inches, 95 inches to 105 inches, 98 inches, 99 inches, 100inches, 101 inches, or 102 inches. In some embodiments, a first impelleris located 15 inches to 20 inches, 16 inches to 19 inches, or 17 inchesto 18 inches from a bottom of the agitation vessel and a second impelleris located 60 inches to 80 inches, 65 inches to 75 inches, 68 inches, 69inches, 70 inches, 71 inches, 72 inches, 73 inches, 74 inches, or 75inches above the first impeller. In some embodiments, a lysed cellcomposition is agitated at least 50 rpm, at least 60 rpm, or at least 70rpm. In some embodiments, a lysed cell composition is agitated at 50 rpmto 70 rpm, 50 rpm to 60 rpm, or 60 rpm to 70 rpm.

In some embodiments, the cell composition, the lysed cell composition,or the lipid are harvested from a vessel by pumping the cellcomposition, the lysed cell composition, or the lipid from the vessel.In some embodiments, the cell composition, the lysed cell composition,or the lipid are harvested from a vessel without agitating the vessel.In some embodiments, the cell composition, the lysed cell composition,or the lipid are harvested from a vessel by pumping, without agitation,the cell composition, the lysed cell composition, or the lipid from thevessel. In some embodiments, the cell composition, the lysed cellcomposition, or the lipid are harvested from a vessel without blowingair. In some embodiments, harvesting the cell composition, the lysedcell composition, or the lipid by the techniques described above resultsin a crude lipid having a low anisidine value (e.g., 26 or less, 25 orless, 20 or less, 15 or less, 10 or less, 5 or less, 2 or less, or 1 orless) and/or a low phosphorus content (e.g., 100 ppm or less, 95 ppm orless, 90 ppm or less, 85 ppm or less, 80 ppm or less, 75 ppm or less, 70ppm or less, 65 ppm or less, 60 ppm or less, 55 ppm or less, 50 ppm orless, 45 ppm or less, 40 ppm or less, 35 ppm or less, 30 ppm or less, 25ppm or less, 20 ppm or less, 15 ppm or less, 10 ppm or less, 5 ppm orless, 4 ppm or less, 3 ppm or less, 2 ppm or less, or 1 ppm or less).

As described herein, the present invention utilizes various combinationsof contacting a lysed cell composition with a first base or raising thepH of a lysed cell composition, contacting a lysed cell composition witha salt, heating a lysed cell composition, and agitating a lysed cellcomposition to provide a treated lysed cell composition. As describedherein, the present invention utilizes various combinations ofcontacting a cell composition with a first base or raising the pH of acell composition, contacting a cell composition with a salt, heating acell composition, and agitating a cell composition to provide a treatedcell composition. The treated cell composition or treated lysed cellcomposition is at least partially demulsified compared with an untreatedcell composition or treated lysed cell composition. Thus, a treated cellcomposition or treated lysed cell composition can be placed in acentrifuge and a lipid can be separated therefrom.

In some embodiments, after raising the pH of a cell composition or lysedcell composition, e.g., by contacting with a first base, the heating thecell composition or lysed cell composition and/or the agitating the cellcomposition or lysed cell composition can decrease the pH of the treatedcell composition or treated lysed cell composition. In order for a lipidto be more effectively separated from a treated cell composition ortreated lysed cell composition by centrifuging, the pH of the treatedcell composition or treated lysed cell composition is raised a secondtime, e.g., by contacting the treated cell composition or the treatedlysed cell composition with a second base. In some embodiments,contacting a treated lysed cell composition with a second base raisesthe pH of the treated cell composition or the treated lysed cellcomposition. In some embodiments, a treated cell composition or atreated lysed cell composition is contacted with a second base to raisethe pH of the treated cell composition or the treated lysed cellcomposition to 7 or above, 7.5 or above, 8 or above, 8.5 or above, 9 orabove, 9.5 or above, 10 or above, 10.5 or above, 11 or above, 11.5 orabove, or 12 or above. In some embodiments, a treated cell compositionor a treated lysed cell composition is contacted with a second base toraise the pH of the treated lysed cell composition to 7 to 13, 7 to 12,7 to 11, 7 to 10, 7 to 9, 7 to 8, 7 to 7.5, 7.5 to 8, 8 to 13, 8 to 12,8 to 11, 8 to 10, 8 to 9, 8 to 8.5, 8.5 to 9, 9 to 12, 9 to 11, 9 to 10,9 to 9.5, 9.5 to 10, 10 to 12, or 10 to 11.

In some embodiments, the pH of a treated cell composition or a treatedlysed cell emulsion is 7 or less, 6 or less, 5 or less, 4 or less, or 3or less.

The processes of the present invention comprise separating a lipid froma treated cell composition or a treated lysed cell composition. In someembodiments, a lipid is separated from a lysed cell emulsion aftercontacting a lysed cell emulsion with a second base, after the agitatinga lysed cell emulsion, or after contacting a lysed cell emulsion with asalt by, for example, permitting the treated lysed cell emulsion to restfor a period of time sufficient for a lipid to separate from the treatedlysed cell emulsion (e.g., as a separate layer). The lipid can besubsequently removed, for example, by decanting, skimming, vacuuming,pumping, sucking off, drawing off, siphoning, or otherwise removing thelipid from the surface of the treated lysed cell emulsion.

In some embodiments, the separating comprises centrifuging a treatedcell composition or a treated lysed cell composition (e.g., at atemperature of 30° C. to 100° C.), whereby the centrifuging separates alipid from the treated cell composition or the treated lysed cellcomposition.

In some embodiments, a process comprises centrifuging a treated cellcomposition or a treated lysed cell composition at a temperature of atleast 10° C., at least 20° C., at least 25° C., at least 30° C., atleast 35° C., at least 40° C., at least 45° C., at least 50° C., atleast 55° C., at least 60° C., at least 65° C., at least 70° C., atleast 75° C., at least 80° C., at least 85° C., at least 90° C., atleast 95° C., or at least 100° C. In some embodiments, a processcomprises centrifuging a treated cell composition or a treated lysedcell composition at a temperature of 10° C. to 100° C., 10° C. to 90°C., 10° C. to 80° C., 20° C. to 100° C., 20° C. to 90° C., 20° C. to 80°C., 25° C. to 100° C., 25° C. to 90° C., 25° C. to 80° C., 25° C. to 75°C., 30° C. to 100° C., 30° C. to 90° C., 30° C. to 80° C., 40° C. to100° C., 40° C. to 90° C., 40° C. to 80° C., 50° C. to 100° C., 50° C.to 90° C., 50° C. to 80° C., 50° C. to 70° C., 60° C. to 100° C., 60° C.to 90° C., 60° C. to 80° C., 60° C. to 70° C., 70° C. to 100° C., or 70°C. to 90° C.

In some embodiments, centrifuging is conducted at a feed rate (of atreated cell composition or a treated lysed cell composition into acentrifuge) of 1 kilogram per minute (kg/min) to 500 kg/min, 1 kg/min to400 kg/min, 1 kg/min to 300 kg/min, 1 kg/min to 200 kg/min, 1 kg/min to100 kg/min, 1 kg/min to 75 kg/min, 1 kg/min to 50 kg/min, 1 kg/min to 40kg/min, 1 kg/min to 30 kg/min, 1 kg/min to 25 kg/min, 1 kg/min to 10kg/min, 10 kg/min to 500 kg/min, 10 kg/min to 400 kg/min, 10 kg/min to300 kg/min, 10 kg/min to 200 kg/min, 10 kg/min to 100 kg/min, 10 kg/minto 75 kg/min, 10 kg/min to 50 kg/min, 10 kg/min to 40 kg/min, 10 kg/minto 30 kg/min, 20 kg/min to 500 kg/min, 20 kg/min to 400 kg/min, 20kg/min to 300 kg/min, 20 kg/min to 200 kg/min, 20 kg/min to 100 kg/min,20 kg/min to 75 kg/min, 20 kg/min to 50 kg/min, 20 kg/min to 40 kg/min,25 kg/min to 500 kg/min, 25 kg/min to 400 kg/min, 25 kg/min to 300kg/min, 25 kg/min to 200 kg/min, 25 kg/min to 100 kg/min, 25 kg/min to75 kg/min, 25 kg/min to 50 kg/min, 30 kg/min to 60 kg/min, 30 kg/min to50 kg/min, 30 kg/min to 40 kg/min, 50 kg/min to 500 kg/min, 100 kg/minto 500 kg/min, or 200 kg/min to 500 kg/min.

The total time required for the separating can vary depending on thevolume of the treated cell composition or the treated lysed cellcomposition. Typical total time for separation (e.g., centrifuge time)is at least 0.1 hour, at least 0.2 hour, at least 0.5 hour, at least 1hour, at least 2 hours, at least 4 hours, at least 6 hours, at least 8hours, at least 10 hours, at least 12 hours, or 0.1 hour to 24 hours,0.5 hour to 24 hours, 1 hour to 12 hours, 2 hours to 10 hours, or 4hours to 8 hours.

In some embodiments, a process of the invention comprises centrifuging atreated cell composition or a treated lysed cell composition at acentrifugal force of 1,000 g to 25,000 g, 1,000 g to 20,000 g, 1,000 gto 10,000 g, 2,000 g to 25,000 g, 2,000 g to 20,000 g, 2,000 g to 15,000g, 3,000 g to 25,000 g, 3,000 g to 20,000 g, 5,000 g to 25,000 g, 5,000g to 20,000 g, 5,000 g to 15,000 g, 5,000 g to 10,000 g, 5,000 g to8,000 g, 10,000 g to 25,000 g, 15,000 g to 25,000 g, or at least 1,000g, at least 2,000, g, at least 4,000 g, at least 5,000 g, at least 7,000g, at least 8,000 g, at least 10,000 g, at least 15,000 g, at least20,000 g, or at least 25,000 g. As used herein, “g” refers to standardgravity or approximately 9.8 m/s². In some embodiments, a process of theinvention comprises centrifuging a treated cell composition or a treatedlysed cell composition at 4,000 rpm to 14,000 rpm, 4,000 rpm to 10,000rpm, 6,000 rpm to 14,000 rpm, 6,000 rpm to 12,000 rpm, 8,000 to 14,000rpm, 8,000 rpm to 12,000 rpm, or 8,000 rpm to 10,000 rpm.

In some embodiments, a process of the invention comprises drying a lipidafter separation of the lipid from a treated cell composition or atreated lysed cell composition in order to remove water from the lipid.In some embodiments, drying the lipid can include, but is not limitedto, heating the lipid to evaporate water. In some embodiments, afterdrying, the lipid has a water content by weight percentage of lipid thatis less than 3%, less than 2.5%, less than 2%, less than 1.5%, less than1%, less than 0.5%, less than 0.1%, or 0%. In some embodiments, afterdrying, the lipid has a water content by weight percentage of lipid of0% to 3%, 0% to 2.5%, 0% to 2%, 0% to 1.5%, 0% to 1%, 0% to 0.5%, 0.1%to 3%, 0.1% to 2.5%, 0.1% to 2%, 0.1% to 1.5%, 0.1% to 1%, 0.1% to 0.5%,0.5% to 3%, 0.5% to 2.5%, 0.5% to 2%, 0.5% to 1.5%, 0.5% to 1%, 1% to3%, 1% to 2.5%, 1% to 2%, 1% to 1.5%, 1.5% to 3%, 1.5% to 2.5%, 1.5% to2%, 2% to 3%, 2% to 2.5%, or 2.5% to 3%.

In some embodiments, a process further comprises refining a lipid by oneor more processes selected from caustic refining, degumming,alkali-refining, bleaching, deodorization, deacidification, or the like,and combinations thereof to remove one or more phospholipids, free fattyacids, phosphatides, color bodies, sterols, odors, and other impurities.As used herein, a “refined oil” is a crude lipid or crude oil that hasbeen refined. As used herein, “a crude lipid” or “a crude oil” is alipid or oil that has not been refined. In some embodiments, the lipidseparated from a demulsified cell composition is a crude lipid.

Various exemplary processes of the present invention are describedschematically in FIGS. 1-4. Referring to FIG. 1, in some embodiments,the present invention is directed to a process (100) for obtaining alipid (110) from a cell (101), comprising lysing (102) the cell (101) toform a lysed cell composition (103). The lysed cell composition iscontacted with a first base (104) to demulsify lysed cell composition(103), contacted with a salt (105) to demulsify lysed cell composition(103), and heated (106), e.g., for 10 minutes to 96 hours, to provide atreated lysed cell composition (107). The treated lysed cell composition(107) is contacted with a second base (108) and separated (109), e.g.,at a temperature of 10° C. to 100° C., to provide a lipid (110).

Referring to FIG. 2, in some embodiments, the present invention isdirected to a process (200) for obtaining a lipid (210) from a cell, theprocess comprising lysing (102) a cell (101) to form a lysed cellcomposition (103). The lysed cell composition is then contacted with abase (204) to demulsify lysed cell composition (103) and to provide atreated lysed cell composition (207). The treated lysed cell composition(207) is separated (209), e.g., at a temperature of 10° C. to 100° C.,to provide a lipid (210).

Referring to FIG. 3, in some embodiments, the present invention isdirected to a process (300) for obtaining a lipid (310) from a cell, theprocess comprising lysing (102) a cell (101) to form a lysed cellcomposition (103). The lysed cell composition is then contacted with asalt (305) to demulsify lysed cell composition (103) and to provide atreated lysed cell composition (307), which is separated (309), e.g., ata temperature of 10° C. to 100° C., to provide a lipid (310).

Referring to FIG. 4, in some embodiments, the present invention isdirected to a process (400) for obtaining a lipid (410) from a cell, theprocess comprising lysing (102) a cell (101) to form a lysed cellcomposition (103). The lysed cell composition is then contacted with asalt (405) to demulsify lysed cell composition (103) and agitated (401),e.g., for 5 minutes to 96 hours, and optionally heated (402), to providea treated lysed cell composition (407). The treated lysed cellcomposition is then separated (409), e.g., at a temperature of 10° C. to100° C., to provide a lipid (410).

In some embodiments, a process of the present invention comprisesconcentrating a broth comprising a microbial cell, a broth comprisingplant material and/or concentrating a lysed cell composition. As usedherein, “concentrating” refers to removing water from a composition.Concentrating can include, but is not limited to, evaporating, chemicaldrying, centrifuging, and the like, and combinations thereof.

In some embodiments, a broth comprising a microbial cell or a brothcomprising plant material is concentrated to provide a lipidconcentration of at least 4%, at least 5%, at least 10%, at least 15%,at least 20%, at least 25%, or at least 30% by weight of the broth. Insome embodiments, a broth comprising a microbial cell or a brothcomprising plant material is concentrated to provide a lipidconcentration of 4% to 40%, 4% to 30%, 4% to 20%, 4% to 15%, 5% to 40%,5% to 30%, 5% to 20%, 10% to 40%, 10% to 30%, 10% to 20%, 15% to 40%,15% to 30%, 20% to 40%, 20% to 30%, 25% to 40%, or 30% to 40% by weightof the broth.

In some embodiments, a cell composition or a lysed cell composition isconcentrated to provide a lipid concentration of at least 4%, at least5%, at least 10%, at least 15%, at least 20%, at least 25%, or at least30% by weight of the lysed cell composition. In some embodiments, a cellcomposition or a lysed cell composition is concentrated to provide alipid concentration of 4% to 40%, 4% to 30%, 4% to 20%, 4% to 15%, 5% to40%, 5% to 30%, 5% to 20%, 10% to 40%, 10% to 30%, 10% to 20%, 15% to40%, 15% to 30%, 20% to 40%, 20% to 30%, 25% to 40%, or 30% to 40% byweight of the lysed cell composition.

In some embodiments, a lipid prepared by a process of the presentinvention has an overall aroma intensity of 2 or less. As used herein,the term “overall aroma intensity” refers to the olfactory sensoryrating given to the lipid by a panel of sensory analysts. As usedherein, the term “sensory analyst” refers to a trained individual thatprovides feedback on and/or rates the sensory characteristics of asubstance.

In some embodiments, a lipid prepared by a process of the presentinvention has an overall aromatic intensity of 3 or less. As usedherein, the term “overall aromatic intensity” refers to the gustatory,or taste, sensory rating given to the lipid by a panel of sensoryanalysts. In some embodiments, the Universal Spectrum descriptiveanalysis method is used to assess the aroma and aromatic characteristicsof samples. This method uses an intensity scale of 0-15, where 0=nonedetected and 15=very high intensity, to measure the aroma and aromaticattributes of the oils.

In some embodiments, a lipid prepared by a process of the presentinvention does not have an aftertaste characterized as fishy. As usedherein, the term “aftertaste” refers to the persistence of a sensationof a flavor in the lipid, as characterized by a panel of sensoryanalysts.

In some embodiments, a process of the present invention provides a crudelipid having a peroxide value (PV) of 5 or less, 4.5 or less, 4 or less,3.5 or less, 3 or less, 2.5 or less, 2 or less, 1.5 or less, 1 or less,0.5 or less, 0.2 or less, or 0.1 or less. As used herein, the terms“peroxide value” or “PV” refer to the measure of primary reactionproducts, such as peroxides and hydroperoxides, that occur duringoxidation of the lipid. In some embodiments, the PV is an indicator ofthe quality of the lipid and the extent of oxidation which has occurredin the lipid having a low PV (i.e., 5 or less) demonstrates increasedstability and sensory profiles than lipids having a PV greater than 5.In some embodiments, adding a base to a lysed cell composition, asdiscussed above, raises the pH of the lysed cell composition andinhibits lipid oxidation, thereby minimizing the amount of free radicalsin the lysed cell composition so that the crude lipid obtained from theprocesses of the invention has a low PV (i.e., 5 or less).

In some embodiments, a process of the present invention provides a crudelipid having an anisidine value (AV) of 26 or less, 25 or less, 20 orless, 15 or less, 10 or less, 5 or less, 2 or less, or 1 or less. Asused herein, the terms “anisidine value” or “AV” refer to the measure ofsecondary reaction products, such as aldehydes and ketones, that occurduring oxidation of the lipid. In some embodiments, the AV is anindicator of the quality of the lipid and the extent of oxidation whichhas occurred in the lipid. A lipid having a low AV (i.e., 26 or less)demonstrates increased stability and sensory profiles than lipids havingan AV greater than 26. In some embodiments, adding a base to a lysedcell composition, as discussed above, raises the pH of the lysed cellcomposition and inhibits lipid oxidation, thereby minimizing the amountof free radicals in the lysed cell composition so that the crude lipidobtained from the processes of the invention has a low AV (i.e., 26 orless).

In some embodiments, a process of the present invention provides a crudelipid having a phosphorus content of 100 ppm or less, 95 ppm or less, 90ppm or less, 85 ppm or less, 80 ppm or less, 75 ppm or less, 70 ppm orless, 65 ppm or less, 60 ppm or less, 55 ppm or less, 50 ppm or less, 45ppm or less, 40 ppm or less, 35 ppm or less, 30 ppm or less, 25 ppm orless, 20 ppm or less, 15 ppm or less, 10 ppm or less, 5 ppm or less, 4ppm or less, 3 ppm or less, 2 ppm or less, or 1 ppm or less.

In some embodiments, a process of the present invention provides a crudelipid that has a lower anisidine value, lower peroxide value, lowerphosphorus content and/or a higher extraction yield than if extractionwas performed using a solvent (e.g., atypical hexane extraction or aFRIOLEX® process (Westfalia Separator AG, Germany)). The FRIOLEX®process which is a process of extracting lipids with a water-solubleorganic solvent as described in U.S. Pat. No. 5,928,696 andInternational Pub. Nos. WO 01/76385 and WO 01/76715, each of which isincorporated by reference herein in its entirety.

In some embodiments, heating the lysed cell composition causes thesecondary reaction products (e.g., aldehydes and ketones) to participatein a reaction similar to the Maillard reaction with proteins present inthe lysed cell composition. The reaction is believed to create productsthat possess antioxidant activity, which reduces the oxidation of thelipid. In some embodiments, additional protein, e.g., soy protein, canbe added to the lysed cell composition to increase the antioxidantactivity. The reduction in oxidation of the lipid reduces the AV of thelipid, reduces any aftertaste of the lipid and/or increases thestability of the lipid. In some embodiments, the stability is increasedat least 5%, at least 10%, at least 15% or at least 20%.

In some embodiments, a lipid extracted by a process of the presentinvention, the biomass remaining after extraction of the lipid, orcombinations thereof can be used directly as a food or food ingredient,such as an ingredient in baby food, infant formula, beverages, sauces,dairy based foods (such as milk, yogurt, cheese and ice-cream), oils(e.g., cooking oils or salad dressings), and baked goods; nutritionalsupplements (e.g., in capsule or tablet forms); feed or feed supplementfor any non-human animal (e.g., those whose products (e.g., meat, milk,or eggs) are consumed by humans); food supplements; and pharmaceuticals(in direct or adjunct therapy application). The term “animal” refers toany organism belonging to the kingdom Animalia and includes any humananimal, and non-human animal from which products (e.g., milk, eggs,poultry meat, beef, pork or lamb) are derived. In some embodiments, thelipid and/or biomass can be used in seafood. Seafood is derived from,without limitation, fish, shrimp and shellfish. The term “products”includes any product derived from such animals, including, withoutlimitation, meat, eggs, milk or other products. When the lipid and/orbiomass are fed to such animals, polyunsaturated lipids can beincorporated into the flesh, milk, eggs or other products of suchanimals to increase their content of these lipids.

Microbial Lipids

In some embodiments, the present invention is directed to a microbiallipid extracted according to the processes of the present invention. Insome embodiments, a crude microbial lipid has an anisidine value of 26or less, 25 or less, 20 or less, 15 or less, 10 or less, 5 or less, 2 orless, or 1 or less, and/or a peroxide value of 5 or less, 4.5 or less, 4or less, 3.5 or less, 3 or less, 2.5 or less, 2 or less, 1.5 or less, 1or less, 0.5 or less, 0.2 or less, or 0.1 or less, and/or a phosphoruscontent of 100 ppm or less, 95 ppm or less, 90 ppm or less, 85 ppm orless, 80 ppm or less, 75 ppm or less, 70 ppm or less, 65 ppm or less, 60ppm or less, 55 ppm or less, 50 ppm or less, 45 ppm or less, 40 ppm orless, 35 ppm or less, 30 ppm or less, 25 ppm or less, 20 ppm or less, 15ppm or less, 10 ppm or less, 5 ppm or less, 4 ppm or less, 3 ppm orless, 2 ppm or less, or 1 ppm or less. In some embodiments, the crudemicrobial lipid has less than 5%, less than 4%, less than 3%, less than2%, or less than 1% by weight or volume of an organic solvent. In someembodiments, the crude microbial lipid has at least 10%, at least 15%,at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, atleast 45%, or at least 50% by weight of a desired PUFA. In someembodiments, the crude microbial lipid has at least 10%, at least 15%,at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, atleast 45%, or at least 50% by weight of DHA, and/or at least 10%, atleast 15%, or at least 20% by weight of DPA n-6, and/or at least 10%, atleast 15%, or at least 20% by weight of EPA, and/or at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, or at least 50% by weight of ARA. In someembodiments a crude microbial lipid extracted according to the processesof the present invention result in a lower anisidine value, lowerperoxide value, lower phosphorus content and/or a higher extractionyield than if extraction was performed using a solvent (e.g., a typicalhexane extraction or a FRIOLEX® process (Westfalia Separator AG,Germany)).

Lipids Extracted From a First Set of Isolated ThraustochytridMicroorganisms

In some embodiments, the present invention is further directed to amicrobial lipid extracted from a thraustochytrid as described in U.S.Pub. No. 2010/0239533 and Int'l. Pub. No. WO 2010/107415, each of whichis incorporated by reference herein in its entirety. In someembodiments, the method comprises growing a thraustochytrid in a cultureto produce a biomass and extracting a lipid comprising omega-3 fattyacids from the biomass. The lipid can be extracted from a freshlyharvested biomass or can be extracted from a previously harvestedbiomass that has been stored under conditions that prevent spoilage.Known methods can be used to culture a thraustochytrid of the invention,to isolate a biomass from the culture, and to analyze the fatty acidprofile of oils extracted from the biomass. See, e.g., U.S. Pat. No.5,130,242, incorporated by reference herein in its entirety. The lipidcan be extracted according to the processes of the present invention.

A microbial lipid of the invention can be any lipid derived from amicroorganism, including, for example: a crude oil extracted from thebiomass of the microorganism without further processing; a refined oilthat is obtained by treating a crude microbial oil with furtherprocessing steps such as refining, bleaching, and/or deodorizing; adiluted microbial oil obtained by diluting a crude or refined microbialoil; or an enriched oil that is obtained, for example, by treating acrude or refined microbial oil with further methods of purification toincrease the concentration of a fatty acid (such as DHA) in the oil.

In some embodiments, the microbial lipid comprises a sterol estersfraction of 0%, at least 0.1%, at least 0.2%, at least 0.5%, at leastabout 1%, at least 1.5%, at least 2%, or at least 5% by weight. In someembodiments, the microbial lipid comprises a sterol esters fraction offrom 0% to 1.5%, 0% to 2%, 0% to 5%, 1% to 1.5%, 0.2% to 1.5%, 0.2% to2%, or 0.2% to 5% by weight. In some embodiments, the microbial lipidcomprises a sterol esters fraction of less than 5%, less than 4%, lessthan 3%, or less than 2% by weight.

In some embodiments, the microbial lipid comprises a triglyceridefraction of at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, or at least 90% by weight. In some embodiments, the microbiallipid comprises a triglyceride fraction of from 65% to 95%, 75% to 95%,or 80% to 95% by weight, or 97% by weight, or 98% by weight.

In some embodiments, the microbial lipid comprises a free fatty acidfraction of at least 0.5%, at least 1%, at least 1.5%, at least 2%, atleast 2.5%, or at least 5% by weight. In some embodiments, the microbiallipid comprises a free fatty acid fraction of from 0.5% to 5%, 0.5% to2.5%, 0.5% to 2%, 0.5% to 1.5%, 0.5% to 1%, 1% to 2.5%, 1% to 5%, 1.5%to 2.5%, 2% to 2.5%, or 2% to 5% by weight. In some embodiments, themicrobial lipid comprises a free fatty acid fraction of less than 5%,less than 4%, less than 3%, less than 2%, or less than 1% by weight.

In some embodiments, the microbial lipid comprises a sterol fraction ofat least 0.5%, at least 1%, at least 1.5%, at least 2%, or at least 5%by weight. In some embodiments, the microbial lipid comprises a sterolfraction of from 0.5% to 1.5%, 1% to 1.5%, 0.5% to 2%, 0.5% to 5%, 1% to2%, or 1% to 5% by weight. In some embodiments, the microbial lipidcomprises a sterol fraction of less than 5%, less than 4%, less than 3%,less than 2%, or less than 1% by weight.

In some embodiments, the microbial lipid comprises a diglyceridefraction of at least 1.5%, at least 2%, at least 2.5%, at least 3%, atleast 3.5%, or at least 5% by weight. In some embodiments, the microbiallipid comprises a diglyceride fraction of from 1.5% to 3%, 2% to 3%,1.5% to 3.5%, 1.5% to 5%, 2.5% to 3%, 2.5% to 3.5%, or 2.5% to 5% byweight.

In some embodiments, the microbial lipid comprises unsaponifiables ofless than 2%, less than 1.5%, less than 1%, or less than 0.5% by weightof the oil.

The lipid classes present in the microbial oil, such as the triglyceridefraction, can be separated by flash chromatography and analyzed by thinlayer chromatography (TLC), or separated and analyzed by other methodsknow in the art.

In some embodiments, the microbial lipid and/or one or more fractionsthereof selected from the triglyceride fraction, the free fatty acidfraction, the sterol fraction, the diglyceride fraction, andcombinations thereof, comprises at least 40%, at least 45%, at least50%, at least 55%, at least 60%, at least 65%, at least 70%, at least75%, or at least 80% by weight DHA. In some embodiments, the microbiallipid and/or one or more fractions thereof selected from thetriglyceride fraction, the free fatty acid fraction, the sterolfraction, the diglyceride fraction, and combinations thereof, comprisesfrom 40% to 45%, 40% to 50%, 40% to 60%, 50% to 60%, 55% to 60%, 40% to65%, 50% to 65%, 55% to 65%, 40% to 70%, 40% to 80%, 50% to 80%, 55% to80%, 60% to 80%, or 70% to 80% by weight DHA. In some embodiments, themicrobial lipid comprises a sterol esters fraction comprising 45% orless, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less,15% or less, or 13% or less by weight DHA. In some embodiments, themicrobial lipid and/or one or more fractions thereof selected from thetriglyceride fraction, the free fatty acid fraction, the sterolfraction, the diglyceride fraction, and combinations thereof, comprises10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less,4% or less, 3% or less, 2% or less, or 1% or less by weight EPA. In someembodiments, the microbial lipid and/or one or more fractions thereofselected from the triglyceride fraction, the free fatty acid fraction,the sterol fraction, the diglyceride fraction, and combinations thereof,comprises from 2% to 3%, 2% to 3.5%, 2.5% to 3.5%, 2% to 6%, 2.5% to 6%,3.0% to 6%, 3.5% to 6%, 5% to 6%, or 2% to 10% by weight EPA. In someembodiments, the microbial lipid and/or one or more fractions thereofselected from the sterol esters fraction, the triglyceride fraction, thefree fatty acid fraction, the sterol fraction, the diglyceride fraction,the polar fraction (including the phospholipid fraction), andcombinations thereof, is substantially free of EPA. In some embodiments,the microbial lipid and/or one or more fractions thereof selected fromthe sterol esters fraction, the triglyceride fraction, the free fattyacid fraction, the sterol fraction, the diglyceride fraction, the polarfraction (including the phospholipid fraction), and combinationsthereof, comprises a weight ratio of DHA to EPA of at least 5:1, atleast 7:1, at least 9:1, at least 10:1, at least 15:1, at least 20:1, atleast 25:1, at least 30:1, or at least 50:1, wherein the microbial lipidand/or one or more fractions thereof comprises 10% or less by weight ofEPA. In some embodiments, the microbial lipid and/or one or morefractions thereof selected from the sterol esters fraction, thetriglyceride fraction, the free fatty acid fraction, the sterolfraction, the diglyceride fraction, the polar fraction (including thephospholipid fraction), and combinations thereof, comprises a weightratio of DHA to EPA of at least 5:1, but less than 20:1. In someembodiments, the weight ratio of DHA to EPA is from 5:1 to 18:1, from7:1 to 16:1, or from 10:1 to 15:1. In some embodiments, the microbiallipid and/or one or more fractions thereof selected from the sterolesters fraction, the triglyceride fraction, the free fatty acidfraction, the sterol fraction, the diglyceride fraction, the polarfraction (including the phospholipid fraction), and combinations thereofcomprises from 0.1% to 0.25%, 0.2% to 0.25%, 0.1% to 0.5%, or 0.1% to1.5% by weight ARA. In some embodiments, the microbial lipid and/or oneor more fractions thereof selected from the sterol esters fraction, thetriglyceride fraction, the free fatty acid fraction, the sterolfraction, the diglyceride fraction, the polar fraction (including thephospholipid fraction), and combinations thereof, comprises 1.5% orless, 1% or less, 0.5% or less, 0.2% or less, or 0.1% or less by weightARA. In some embodiments, the microbial lipid and/or one or morefractions thereof selected from the sterol esters fraction, thetriglyceride fraction, the free fatty acid fraction, the sterolfraction, the diglyceride fraction, the polar fraction (including thephospholipid fraction), and combinations thereof, is substantially freeof ARA. In some embodiments, the microbial lipid and/or one or morefractions thereof selected from the sterol esters fraction, thetriglyceride fraction, the free fatty acid fraction, the diglyceridefraction, the polar fraction (including the phospholipid fraction), andcombinations thereof, comprises a weight ratio of DHA to ARA of at least20:1, at least 30:1, at least 35:1, at least 40:1, at least 60:1, atleast 80:1, at least 100:1, at least 150:1, at least 200:1, at least250:1, or at least 300:1. In some embodiments, the microbial lipidand/or one or more fractions thereof selected from the sterol estersfraction, the triglyceride fraction, the free fatty acid fraction, thesterol fraction, the diglyceride fraction, the polar fraction (includingthe phospholipid fraction), and combinations thereof, comprises from0.5% to 1%, 0.5% to 2%, 0.5% to 2.5%, 0.5% to 3%, 0.5% to 3.5%, 0.5% to5%, 0.5% to 6%, 1% to 2%, 2% to 3%, 2% to 3.5%, 1% to 2.5%, 1% to 3%, 1%to 3.5%, 1% to 5%, or 1% to 6% by weight DPA n-6. In some embodiments,the microbial lipid and/or one or more fractions thereof selected fromthe sterol esters fraction, the triglyceride fraction, the free fattyacid fraction, the sterol fraction, the diglyceride fraction, the polarfraction (including the phospholipid fraction), and combinationsthereof, comprises 6% or less, 5% or less, 3% or less, 2.5% or less, 2%or less, 1% or less, or 0.5% or less by weight DPA n-6. In someembodiments, the microbial lipid and/or one or more fractions thereofselected from the sterol esters fraction, the triglyceride fraction, thefree fatty acid fraction, the sterol fraction, the diglyceride fraction,the polar fraction (including the phospholipid fraction), andcombinations thereof, is substantially free of DPA n-6. In someembodiments, the microbial lipid and/or one or more fractions thereofselected from the sterol esters fraction, the triglyceride fraction, thefree fatty acid fraction, the sterol fraction, the diglyceride fraction,the polar fraction (including the phospholipid fraction), andcombinations thereof, comprises a weight ratio of DHA to DPA n-6 ofgreater than 6:1, of at least 8:1, at least 10:1, at least 15:1, atleast 20:1, at least 25:1, at least 50:1, or at least 100:1. In someembodiments, the microbial lipid and/or one or more fractions thereofselected from the sterol esters fraction, the triglyceride fraction, thefree fatty acid fraction, the sterol fraction, the diglyceride fraction,the polar fraction (including the phospholipid fraction), andcombinations thereof, comprises 5% or less, 4% or less, 3% or less, 2%or less, 1.5% or less, 1% or less, or 0.5% or less by weight each oflinoleic acid (18:2 n-6), linolenic acid (18:3 n-3), eicosenoic acid(20:1 n-9), and erucic acid (22:1 n-9). In some embodiments, themicrobial lipid and/or one or more fractions thereof selected from thesterol esters fraction, the triglyceride fraction, the free fatty acidfraction, the sterol fraction, the diglyceride fraction, the polarfraction (including the phospholipid fraction), and combinationsthereof, comprises 5% or less, 4% or less, 3% or less, 2% or less, 1.5%or less, or 1% or less by weight of heptadecanoic acid (17:0). In someembodiments, the microbial lipid and/or one or more fractions thereofcomprise 0.01% to 5% by weight, 0.05% to 3% by weight, or 0.1% to 1% byweight of heptadecanoic acid.

In some embodiments, an extracted microbial lipid comprises atriglyceride fraction of at least 70% by weight, wherein thedocosahexaenoic acid content of the triglyceride fraction is at least50% by weight, wherein the docosapentaenoic acid n-6 content of thetriglyceride fraction is from at least 0.5% by weight to 6% by weight,and wherein the oil has an anisidine value of 26 or less. In someembodiments, an extracted microbial lipid comprises a triglyceridefraction of at least 70% by weight, wherein the docosahexaenoic acidcontent of the triglyceride fraction is at least 40% by weight, whereinthe docosapentaenoic acid n-6 content of the triglyceride fraction isfrom at least 0.5% by weight to 6% by weight, wherein the ratio ofdocosahexaenoic acid to docosapentaenoic acid n-6 is greater than 6:1,and wherein the lipid has an anisidine value of 26 or less. In someembodiments, an extracted microbial lipid comprises a triglyceridefraction of at least 70% by weight, wherein the docosabexaenoic acidcontent of the triglyceride fraction is at least 60% by weight andwherein the lipid has an anisidine value of 26 or less. In someembodiments, an extracted microbial lipid having any of the above fattyacid profiles has an anisidine value of 26 or less, 25 or less, 20 orless, 15 or less, 10 or less, 5 or less, 2 or less, or 1 or less and/ora peroxide value of 5 or less, 4.5 or less, 4 or less, 3.5 or less, 3 orless, 2.5 or less, 2 or less, 1.5 or less, 1 or less, 0.5 or less, 0.2or less, or 0.1 or less, and/or a phosphorus content of 100 ppm or less,95 ppm or less, 90 ppm or less, 85 ppm or less, 80 ppm or less, 75 ppmor less, 70 ppm or less, 65 ppm or less, 60 ppm or less, 55 ppm or less,50 ppm or less, 45 ppm or less, 40 ppm or less, 35 ppm or less, 30 ppmor less, 25 ppm or less, 20 ppm or less, 15 ppm or less, 10 ppm or less,5 ppm or less, 4 ppm or less, 3 ppm or less, 2 ppm or less, or 1 ppm orless. In some embodiments, an extracted microbial lipid having any ofthe above fatty acid profiles is extracted from an isolatedthraustochytrid microorganism having the characteristics of thethraustochytrid species deposited under ATCC Accession No. PTA-9695,PTA-9696, PTA-9697, or PTA-9698. In some embodiments, an extractedmicrobial lipid having any of the above fatty acid profiles is a crudelipid. In some embodiments, the crude lipid has less than 5% by weightor volume of an organic solvent. In some embodiments the microbial lipidextracted according to the processes of the present invention result ina lower anisidine value, lower peroxide value, lower phosphorus contentand/or a higher extraction yield if extraction was performed using asolvent (e.g., atypical hexane extraction or a FRIOLEX® process(Westfalia Separator AG, Germany)).

Lipids Extracted from a Second Set of Isolated ThraustochytridMicroorganisms

In some embodiments, the present invention is further directed to amicrobial lipid extracted from a thraustochytrid as described in U.S.application Ser. No. 12/729,013 and PCT/US2010/028175, each of which isincorporated by reference herein in its entirety. In some embodiments,the method comprises growing a thraustochytrid in a culture to produce abiomass and extracting a lipid comprising omega-3 fatty acids from thebiomass. The lipid can be extracted from a freshly harvested biomass orcan be extracted from a previously harvested biomass that has beenstored under conditions that prevent spoilage. Known methods can be usedto culture a thraustochytrid of the invention, to isolate a biomass fromthe culture, and to analyze the fatty acid profile of oils extractedfrom the biomass. See, e.g., U.S. Pat. No. 5,130,242, incorporated byreference herein in its entirety. The lipid can be extracted accordingto the processes of the present invention.

A microbial lipid of the invention can be any lipid derived from amicroorganism, including, for example: a crude oil extracted from thebiomass of the microorganism without further processing; a refined oilthat is obtained by treating a crude microbial oil with furtherprocessing steps such as refining, bleaching, and/or deodorizing; adiluted microbial oil obtained by diluting a crude or refined microbialoil; or an enriched oil that is obtained, for example, by treating acrude or refined microbial oil with further methods of purification toincrease the concentration of a fatty acid (such as DHA) in the oil.

In some embodiments, the microbial lipid comprises a sterol estersfraction of 0%, at least 0.1%, at least 0.2%, at least 0.5%, at least1%, at least 1.5%, at least 2%, or at least 5% by weight. In someembodiments, the microbial lipid comprises a sterol esters fraction of0% to 1.5%, 0% to 2%, 0% to 5%, 1% to 1.5%, 0.2% to 1.5%, 0.2% to 2%, or0.2% to 5% by weight. In some embodiments, the microbial lipid comprisesa sterol esters fraction of 5% or less, 4% or less, 3% or less, 2% orless, 1% or less, 0.5% or less, 0.3% or less, 0.2% or less, 0.5% orless, 0.4% or less, 0.3% or less, or 0.2% or less by weight.

In some embodiments, the microbial lipid comprises a triacylglycerolfraction of at least 35%, at least 40%, at least 45%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, or at least 90% by weight. In some embodiments,the microbial lipid comprises a triacylglycerol fraction of 35% to 98%,35% to 90%, 35% to 80%, 35% to 70%, 35% to 70%, 35% to 65%, 40% to 70%,40% to 65%, 40% to 55%, 40% to 50%, 65% to 95%, 75% to 95%, 75% to 98%,80% to 95%, 80% to 98%, 90% to 96%, 90% to 97%, 90% to 98%, 90%, 95%,97%, or 98% by weight.

In some embodiments, the microbial lipid comprises a diacylglycerolfraction of at least 10%, at least 11%, at least 12%, at least 13%, atleast 14%, at least 15%, at least 16%, at least 17%, at least 18%, atleast 19%, or at least 20% by weight. In some embodiments, the microbiallipid comprises a diacylglycerol fraction of 10% to 45%, 10% to 40%, 10%to 35%, 10% to 30%, 15% to 40%, 15% to 35%, or 15% to 30% by weight. Insome embodiments, the microbial lipid comprises a 1,2-diacylglycerolfraction of at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%,at least 1%, at least 5%, at least 10%, at least 11%, at least 12%, atleast 13%, at least 14%, at least 15%, at least 16%, at least 17%, atleast 18%, at least 19%, or at least 20% by weight. In some embodiments,the microbial lipid comprises a diacylglycerol fraction of 0.2% to 45%,0.2% to 30%, 0.2% to 20%, 0.2% to 10%, 0.2% to 5%, 0.2% to 1%, 0.2% to0.8%, 0.4% to 45%, 0.4% to 30%, 0.4% to 20%, 0.4% to 10%, 0.4% to 5%,0.4% to 1%, 0.4% to 0.8%, 0.5% to 1%, 0.5% to 0.8%, 10% to 45%, 10% to40%, 10% to 35%, 10% to 30%, 15% to 40%, 15% to 35%, 15% to 30%, or 15%to 25% by weight. In some embodiments, the microbial lipid comprises a1,3-diacylglycerol fraction of at least 0.1%, at least 0.2%, at least0.5%, at least 1%, at least 2%, at least 2.5%, or at least 3% by weight.

In some embodiments, the microbial lipid comprises a sterol fraction ofat least 0.3%, at least 0.4%, at least 0.5%, at least 1%, at least 1.5%,at least 2%, or at least 5% by weight. In some embodiments, themicrobial lipid comprises a sterol fraction of 0.3% to 5%, 0.3% to 2%,0.3% to 1.5%, 0.5% to 1.5%, 1% to 1.5%, 0.5% to 2%, 0.5% to 5%, 1% to2%, or 1% to 5% by weight. In some embodiments, the microbial lipidcomprises a sterol fraction of 5% or less, 4% or less, 3% or less, 2% orless, 1.5% or less, or 1% or less by weight.

In some embodiments, the microbial lipid comprises a phospholipidfraction of at least 2%, at least 5%, or at least 8% by weight. In someembodiments, the microbial lipid comprises a phospholipid fraction of 2%to 25%, 2% to 20%, 2% to 15%, 2% to 10%, 5% to 25%, 5% to 20%, 5% to20%, 5% to 10%, or 7% to 9% by weight. In some embodiments, themicrobial lipid comprises a phospholipid fraction of less than 20%, lessthan 15%, less than 10%, less than 9%, or less than 8% by weight. Insome embodiments, the microbial lipid is substantially free ofphospholipids.

In some embodiments, the microbial lipid comprises unsaponifiables ofless than 2%, less than 1.5%, less than 1%, or less than 0.5% by weightof the oil.

The lipid classes present in the microbial lipid, such as atriacylglycerol fraction, can be separated by flash chromatography andanalyzed by thin layer chromatography (TLC), or separated and analyzedby other methods known in the art.

In some embodiments, the microbial lipid and/or one or more fractionsthereof selected from the triacylglycerol fraction, the free fatty acidfraction, the sterol fraction, the diacylglycerol fraction, andcombinations thereof, comprises at least 5%, at least 10%, more than10%, at least 12%, at least 13%, at least 14%, at least 15%, at least16%, at least 17%, at least 18%, at least 19%, at least 20%, at least25%, at least 30%, least 35%, at least 40%, or at least 45% by weightEPA. In some embodiments, the microbial lipid and/or one or morefractions thereof selected from the triacylglycerol fraction, the freefatty acid fraction, the sterol fraction, the diacylglycerol fraction,and combinations thereof, comprises 5% to 55%, 5% to 50%, 5% to 45%, 5%to 40%, 5% to 35%, 5% to 30%, 10% to 55%, 10% to 50%, 10% to 45%, 10% to40%, 10% to 35%, 10% to 30%, at least 12% to 55%, at least 12% to 50%,at least 12% to 45%, at least 12% to 40%, at least 12% to 35%, or atleast 12% to 30%, 15% to 55%, 15% to 50%, 15% to 45%, 15% to 40%, 15% to35%, 15% to 30%, 15% to 25%, 15% to 20%, 20% to 55%, 20% to 50%, 20% to45%, 20% to 40%, or 20% to 30% by weight EPA. In some embodiments, themicrobial lipid and/or one or more fractions thereof selected from thetriacylglycerol fraction, the diacylglycerol fraction, the sterolfraction, the sterol esters fraction, the free fatty acids fraction, thephospholipid fraction, and combinations thereof, comprises at least 5%,at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, at least 50%, or at least 60% by weight DHA. Insome embodiments, the microbial lipid and/or one or more fractionsthereof selected from the triacylglycerol fraction, the diacylglycerolfraction, the sterol fraction, the sterol esters fraction, the freefatty acids fraction, the phospholipid fraction, and combinationsthereof, comprises 5% to 60%, 5% to 55%, 5% to 50%, 5% to 40%, 10% to60%, 10% to 50%, 10% to 40%, 20% to 60%, 25% to 60%, 25% to 50%, 25% to45%, 30% to 50%, 35% to 50%, or 30% to 40% by weight DHA. In someembodiments, the microbial lipid and/or one or more fractions thereofselected from the triacylglycerol fraction, the diacylglycerol fraction,the sterol fraction, the sterol esters fraction, the free fatty acidsfraction, the phospholipid fraction, and combinations thereof, comprises10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less,4% or less, 3% or less, 2% or less, or 1% or less by weight DHA. In someembodiments, the microbial lipid and/or one or more fractions thereofselected from the triacylglycerol fraction, the diacylglycerol fraction,the sterol fraction, the sterol esters fraction, the free fatty acidsfraction, the phospholipid fraction, and combinations thereof, comprises1% to 10%, 1% to 5%, 2% to 5%, 3% to 5%, or 3% to 10% by weight of thefatty acids as DHA. In some embodiments, the microbial lipid and/or oneor more fractions thereof selected from the triacylglycerol fraction,the diacylglycerol fraction, the sterol fraction, the sterol estersfraction, the free fatty acids fraction, the phospholipid fraction, andcombinations thereof, is substantially free of DHA. In some embodiments,the microbial lipid and/or one or more fractions thereof selected fromthe triacylglycerol fraction, the diacylglycerol fraction, the sterolfraction, the sterol esters fraction, the free fatty acids fraction, thephospholipid fraction, and combinations thereof, comprises 0.1% to 5%,0.1% to less than 5%, 0.1% to 4%, 0.1% to 3%, 0.1% to 2%, 0.2% to 5%,0.2% to less than 5%, 0.2% to 4%, 0.2% to 3%, 0.2% to 2%, 0.3% to 2%,0.1% to 0.5%, 0.2% to 0.5%, 0.1% to 0.4%, 0.2% to 0.4%, 0.5% to 2%, 1%to 2%, 0.5% to 1.5%, or 1% to 1.5% by weight ARA. In some embodiments,the microbial lipid and/or one or more fractions thereof selected fromthe triacylglycerol fraction, the diacylglycerol fraction, the sterolfraction, the sterol esters fraction, the free fatty acids fraction, thephospholipid fraction, and combinations thereof, comprises 5% or less,less than 5%, 4% or less, 3% or less, 2% or less, 1.5% or less, 1% orless, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, or 0.1% orless by weight ARA. In some embodiments, the microbial lipid and/or oneor more fractions thereof selected from the triacylglycerol fraction,the diacylglycerol fraction, the sterol fraction, the sterol estersfraction, the free fatty acids fraction, the phospholipid fraction, andcombinations thereof, is substantially free of ARA. In some embodiments,the microbial lipid and/or one or more fractions thereof selected fromthe triacylglycerol fraction, the diacylglycerol fraction, the sterolfraction, the sterol esters fraction, the free fatty acids fraction, thephospholipid fraction, and combinations thereof, comprises 0.4% to 2%,0.4% to 3%, 0.4% to 4%, 0.4% to 5%, 0.4% to less than 5%, 0.5% to 1%,0.5% to 2%, 0.5% to 3%, 0.5% to 4%, 0.5% to 5%, 0.5% to less than 5%, 1%to 2%, 1% to 3%, 1% to 4%, 1% to 5%, or 1% to less than 5% by weight DPAn-6. In some embodiments, the microbial lipid and/or one or morefractions thereof selected from the triacylglycerol fraction, thediacylglycerol fraction, the sterol fraction, the sterol estersfraction, the free fatty acids fraction, the phospholipid fraction, andcombinations thereof, comprises 5%, less than 5%, 4% or less, 3% orless, 2% or less, 1% or less, 0.75% or less, 0.6% or less, or 0.5% orless by weight DPA n-6. In some embodiments, the microbial lipid and/orone or more fractions thereof selected from the triacylglycerolfraction, the diacylglycerol fraction, the sterol fraction, the sterolesters fraction, the free fatty acids fraction, the phospholipidfraction, and combinations thereof, is substantially free of DPA n-6. Insome embodiments, the microbial lipid and/or one or more fractionsthereof selected from the triacylglycerol fraction, the diacylglycerolfraction, the sterol fraction, the sterol esters fraction, the freefatty acids fraction, the phospholipid fraction, and combinationsthereof, comprises fatty acids with 5% or less, less than 5%, 4% orless, 3% or less, or 2% or less by weight of oleic acid (18:1 n-9),linoleic acid (18:2 n-6), linolenic acid (18:3 n-3), eicosenoic acid(20:1 n-9), erucic acid (22:1 n-9), stearidonic acid (18:4 n-3), orcombinations thereof.

In some embodiments, an extracted microbial lipid comprises at least 20%by weight eicosapentaenoic acid and less than 5% by weight each ofarachidonic acid, docosapentaenoic acid n-6, oleic acid, linoleic acid,linolenic acid, eicosenoic acid, erucic acid, and stearidonic acid. Insome embodiments, an extracted microbial lipid comprises atriacylglycerol fraction of at least 10% by weight, wherein at least 12%by weight of the fatty acids in the triacylglycerol fraction iseicosapentaenoic acid, wherein at least 25% by weight of the fatty acidsin the triacylglycerol fraction is docosahexaenoic acid, and whereinless than 5% by weight of the fatty acids in the triacylglycerolfraction is arachidonic acid. In some embodiments, an extractedmicrobial lipid having any of the above fatty acid profiles has ananisidine value of 26 or less, 25 or less, 20 or less, 15 or less, 10 orless, 5 or less, 2 or less, or 1 or less, and/or a peroxide value of 5or less, 4.5 or less, 4 or less, 3.5 or less, 3 or less, 2.5 or less, 2or less, 1.5 or less, 1 or less, 0.5 or less, 0.2 or less, or 0.1 orless, and/or a phosphorus content of 100 ppm or less, 95 ppm or less, 90ppm or less, 85 ppm or less, 80 ppm or less, 75 ppm or less, 70 ppm orless, 65 ppm or less, 60 ppm or less, 55 ppm or less, 50 ppm or less, 45ppm or less, 40 ppm or less, 35 ppm or less, 30 ppm or less, 25 ppm orless, 20 ppm or less, 15 ppm or less, 10 ppm or less, 5 ppm or less, 4ppm or less, 3 ppm or less, 2 ppm or less, or 1 ppm or less. In someembodiments, an extracted microbial lipid having any of the above fattyacid profiles is extracted from an isolated thraustochytridmicroorganism having the characteristics of the thraustochytrid speciesdeposited under ATCC Accession No. PTA-10208, PTA-10209, PTA-10210,PTA-10211, PTA-10212, PTA-10213, PTA-10214, or PTA-10215. In someembodiments, an extracted microbial lipid having any of the above fattyacid profiles is a crude lipid. In some embodiments, the crude lipid hasless than 5% by weight or volume of an organic solvent. In someembodiments a microbial lipid extracted according to the processes ofthe present invention result in a lower anisidine value, and/or lowerperoxide value, and/or lower phosphorus content than if extraction wasperformed using a typical hexane extraction or a FRIOLEX® process(Westfalia Separator AG, Germany).

In some embodiments, a lipid obtained by any of the processes of thepresent invention comprises at least 20% by weight eicosapentaenoic acidand less than 5% by weight each of arachidonic acid, docosapentaenoicacid n-6, oleic acid, linoleic acid, linolenic acid, eicosenoic acid,erucic acid, and stearidonic acid. In some embodiments, a lipid obtainedby any of the processes of the present invention comprises atriacylglycerol fraction of at least 10% by weight, wherein at least 12%by weight of the fatty acids in the triacylglycerol fraction iseicosapentaenoic acid, wherein at least 25% by weight of the fatty acidsin the triacylglycerol fraction is docosahexaenoic acid, and whereinless than 5% by weight of the fatty acids in the triacylglycerolfraction is arachidonic acid. In some embodiments, a lipid obtained byany of the processes of the present invention having any of the abovefatty acid profiles has an anisidine value of 26 or less, 25 or less, 20or less, 15 or less, 10 or less, 5 or less, or 2 or less, or 1 or lessand/or a peroxide value of 5 or less, 4.5 or less, 4 or less, 3.5 orless, 3 or less, 2.5 or less, 2 or less, 1.5 or less, 1 or less, 0.5 orless, 0.2 or less, or 0.1 or less, and/or a phosphorus content of 100ppm or less, 95 ppm or less, 90 ppm or less, 85 ppm or less, 80 ppm orless, 75 ppm or less, 70 ppm or less, 65 ppm or less, 60 ppm or less, 55ppm or less, 50 ppm or less, 45 ppm or less, 40 ppm or less, 35 ppm orless, 30 ppm or less, 25 ppm or less, 20 ppm or less, 15 ppm or less, 10ppm or less, 5 ppm or less, 4 ppm or less, 3 ppm or less, 2 ppm or less,or 1 ppm or less. In some embodiments, a lipid obtained by any of theprocesses of the present invention having any of the above fatty acidprofiles is extracted from an isolated thraustochytrid microorganismhaving the characteristics of the thraustochytrid species depositedunder ATCC Accession No. PTA-10208, PTA-10209, PTA-10210, PTA-10211,PTA-10212, PTA-10213, PTA-10214, or PTA-10215. In some embodiments, alipid obtained by any of the processes of the present invention havingany of the above fatty acid profiles is a crude lipid. In someembodiments, the crude lipid has less than 5% by weight or volume of anorganic solvent. In some embodiments a lipid extracted according to theprocesses of the present invention result in a lower anisidine value,lower peroxide value, lower phosphorus content and/or a higherextraction yield if extraction was performed using a solvent (e.g.,atypical hexane extraction or a FRIOLEX® process (Westfalia SeparatorAG, Germany)).

Lipids Extracted from an Isolated Microorganism of the SpeciesCryptiecodinium Cohnii

In some embodiments, the present invention is further directed to acrude lipid extracted from a microorganism of the speciesCrypthecodinium cohnii. In some embodiments, the method comprisesgrowing a microorganism of the species Crypthecodinium cohnii in aculture to produce a biomass and extracting a lipid comprising omega-3fatty acids from the biomass. The lipid can be extracted from a freshlyharvested biomass or can be extracted from a previously harvestedbiomass that has been stored under conditions that prevent spoilage.Known methods can be used to culture a microorganism of the speciesCrypthecodinium cohnii, and to isolate a biomass from the culture. See,e.g., U.S. Pat. No. 7,163,811, incorporated by reference herein in itsentirety. The lipid can be extracted according to the processes of thepresent invention.

In some embodiments, the crude lipid extracted from a microorganism ofthe species Crypthecodinium cohnii according to the extraction methodsof the present invention can have a lower phosphorus content compared tousing a typical hexane extraction method. In some embodiments, the crudelipid extracted from a microorganism of the species Crypthecodiniumcohnii comprises a phosphorus content of 100 ppm or less, 95 ppm orless, 90 ppm or less, 85 ppm or less, 80 ppm or less, 75 ppm or less, 70ppm or less, 65 ppm or less, 60 ppm or less, 55 ppm or less, 50 ppm orless, 45 ppm or less, 40 ppm or less, 35 ppm or less, 30 ppm or less, 25ppm or less, 20 ppm or less, 15 ppm or less, 10 ppm or less, 5 ppm orless, 4 ppm or less, 3 ppm or less, 2 ppm or less, or 1 ppm or less. Insome embodiments, the crude oil has an anisidine value of 26 or less, 25or less, 20 or less, 15 or less, 10 or less, 5 or less, or 2 or less, or1 or less and/or a peroxide value of 5 or less, 4.5 or less, 4 or less,3.5 or less, 3 or less, 2.5 or less, 2 or less, 1.5 or less, 1 or less,0.5 or less, 0.2 or less, or 0.1 or less. In some embodiments a crudemicrobial lipid extracted according to the processes of the presentinvention result in a lower anisidine value, lower peroxide value, lowerphosphorus content and/or a higher extraction yield if extraction wasperformed using a solvent (e.g., atypical hexane extraction or aFRIOLEX® process (Westfalia Separator AG, Germany)).

Having generally described the invention, a further understanding can beobtained by reference to the examples provided herein. These examplesare given for purposes of illustration only and are not intended to belimiting. The following examples are illustrative, but not limiting, ofa process and a lipid prepared by a process of the present invention.Other suitable modifications and adaptations of the variety ofconditions and parameters normally encountered in extraction of a lipidfrom a cell, and which would become apparent to those skilled in theart, and are within the spirit and scope of the invention.

EXAMPLES Example 1

A cell broth (20,000 kg) containing microbial cells (Schizochytrium) washeated to 60° C. Enzymes (i.e., Alcalase 2.4 L FG 0.5%) were added tothe cell biomass to lyse the cells and form an emulsified lysed cellcomposition. The emulsified lysed cell composition was first treatedwith a first base (NaOH, 250 kg of 50% w/w solution) until the pH of thelysed cell composition was from 10.4 to 10.6. Next, a salt (solid NaCl,in an amount of 2%, by weight, of the lysed cell composition) was addedto the lysed cell composition. The lysed cell composition was thenheated to a temperature of 85° C. to 102° C. and held at thattemperature level for 24 hours to 70 hours. A second base (NaOH, 50% w/wsolution, 40 kg) was then added to the lysed cell composition until thepH was above 8. The lysed cell composition was then centrifuged toseparate the lysed cell composition into three phases: a top phasecontaining a lipid layer, a middle phase containing an emulsion layer,and a bottom phase containing a solid layer. The lysed cell compositionwas then centrifuged at 40° C. to 80° C. using a Westfalia RSE110Centrifuge (Westfalia Separator Industry GmbH, Germany), operating at6,000 rpms at a feed rate of 30 kg/min to separate a lipid from thelysed cell composition. The centrifuging provided three phases: an upperphase containing a lipid, a middle phase containing an emulsion, and abottom phase containing a solid/liquid emulsion. The pH of the lysedcell composition was maintained at 7.5 to 8.5 during the centrifuging.The total time to centrifuge the entire 20,000 kg batch wasapproximately 10 to 11 hours. The lipid layer was separated and had amoisture content of approximately 1% by weight.

Example 2

A cell broth (500 g) containing microbial cells (Crypthecodinium cohnii)was concentrated from approximately 7% biomass to 13.5% biomass, byweight of the broth. The broth was homogenized at a pressure of 10,000psi (2 passes) to form a lysed cell composition. The lysed cellcomposition was treated with a base (i.e., NaOH, 10 g of a 50% w/wsolution) until the pH of the lysed cell composition was 10.4 to 10.6. Asalt (solid NaCl, in an amount of 2% by weight of the lysed cellcomposition) was added to the lysed cell composition. The lysed cellcomposition was then heated to a temperature of 85° C. to 92° C. andheld at that temperature range for 15 minutes to 2 hours. The lysed cellcomposition was then centrifuged at a temperature of 70° C. to 90° C.using a Bench Top Sigma 6K15 Centrifuge (SIGMA Laborzentrifugen GmbH,Germany), operating at 5,400 rpm to separate the lysed cell compositioninto three phases: an upper phase containing a lipid, a middle phasecontaining an emulsion, and a bottom phase containing a solid/liquidemulsion. The pH of the lysed cell composition during centrifuging wasmaintained at 6.5 to 8.5. The total time to centrifuge was 5 minutes.The lipid layer was separated and had a moisture content ofapproximately 1% by weight.

Example 3

A cell broth (20,000 kg) containing microbial cells (Schizochytrium) washeated to 60° C. Enzymes (i.e., Alcalase 2.4 L FG 0.5%) were added tothe cell biomass to lyse the cells and form a lysed cell composition.Next, a salt (solid Na₂SO₄, 2,000 kg, or 10%, by weight, of the lysedcell composition) was added to the lysed cell composition. The lysedcell composition was then agitated for 24 hours to 48 hours at roomtemperature. The lysed cell composition was then centrifuged at 40° C.to 75° C. using a Westfalia RSE110 Centrifuge (Westfalia SeparatorIndustry GmbH, Germany), operating at 6,000 rpm at a feed rate of 40kg/min to separate a lipid from the lysed cell composition. Thecentrifuging provided three phases: an upper phase containing a lipid, amiddle phase containing an emulsion, and a bottom phase containing asolid/liquid emulsion. The total time to centrifuge the entire 20,000 kgbatch was approximately 8 to 9 hrs. The lipid layer was separated fromthe centrifuged lysed cell composition.

Example 4

A pasteurized cell broth (500 g) containing microbial cells (ATCCAccession No. PTA-10208) was provided. Enzymes (i.e., Alcalase 2.4 L FG0.5%) were added to the cell biomass to lyse the cells and form anemulsified lysed cell composition. The emulsified lysed cell compositionwas treated with a first base (i.e., a 25% solution of NaOH) to adjustthe pH of the lysed cell composition to 10.5. Next, a salt (solid NaCl,in an amount of 2%, by weight, of the lysed cell composition) was addedto the lysed cell composition. The lysed cell composition was thenheated to a temperature of 95° C. and held at that temperature level for2 hours while agitating the lysed cell composition. A second base (i.e.,a 25% solution of NaOH) was then added to the lysed cell compositionuntil the pH was 8.3. The lysed cell composition was then centrifuged at5,100 rpm for 5 minutes to separate the lysed cell composition and yielda lipid layer and a small emulsion layer.

Example 5

A cell broth (500 g) that was concentrated and pasteurized containingmicrobial cells (ATCC Accession No. PTA-9695) was provided. Enzymes(i.e., Alcalase 2.4 L FG 0.5%) were added to the cell biomass to lysethe cells and form an emulsified lysed cell composition. The emulsifiedlysed cell composition was treated with a base (i.e., a 25% solution ofNaOH) to adjust the pH of the lysed cell composition to 10.5. Next, asalt (solid NaCl, in an amount of 2%, by weight, of the lysed cellcomposition) was added to the lysed cell composition. The lysed cellcomposition was then heated to a temperature of 95° C. and held at thattemperature level for 1 hour while agitating the lysed cell compositionand the pH dropped to 8.5. After an hour in the fermentation brothhaving a total of 10 ml, there was an about 1 ml layer of oil (lipid)and an about 6 ml layer of emulsion. The lysed cell composition washeated for a total of 220 minutes and the emulsion layer started todisappear. The lysed cell composition was then centrifuged at 5,100 rpmfor 5 minutes to separate the lysed cell composition. The extractionyield of the lipid was 58.8 by weight %. The anisidine value (AV) of thecrude oil was 11.3. The cell breakage yield was in a range of 93% to 95%by weight.

Example 6

A pasteurized cell broth (473 g) containing microbial cells of theisolated thraustochytrid deposited under ATCC Accession No. PTA-9695 wasprovided. Enzymes (i.e., Alcalase 2.4 L FG 0.5%) were added to the cellbiomass to lyse the cells and form an emulsified lysed cell composition.The emulsified lysed cell composition was treated with a first base(i.e., a 25% solution of NaOH) to adjust the pH of the lysed cellcomposition to 10.62. Next, a salt (solid NaCl, in an amount of 2%, byweight, of the lysed cell composition) was added to the lysed cellcomposition. The lysed cell composition was then heated to a temperatureof 95° C. and held at that temperature level for 3 hours while agitatingthe lysed cell composition. A second base (i.e., a 25% caustic solutionof NaOH) was then added to the lysed cell composition until the pH was8.13. The lysed cell composition was then centrifuged at 5,100 rpm for 5minutes to separate the lysed cell composition and yield a lipid layerand an emulsion layer in equal amounts. In order to determine if raisingthe pH increased the yield of the lipid layer, additional second base(i.e., a 25% solution of NaOH) was added to the separated lysed cellcomposition until the pH was 9.02 and the lysed cell composition wasagain centrifuged at 5,100 rpm for 5 minutes. This resulted in a similaryield of lipid layer. Additional second base was added again to theseparated lysed cell composition until the pH was 10.12 and the lysedcell composition was again centrifuged at 5,100 rpm for 5 minutes.Again, this resulted in a similar yield of lipid layer.

Example 7

A pasteurized cell broth (470 g) containing microbial cells (ATCCAccession No. PTA-9695) was provided. The cell biomass was mechanicallyhomogenized to lyse the cells and form an emulsified lysed cellcomposition. The emulsified lysed cell composition was treated with afirst base (i.e., a 25% solution of NaOH) to adjust the pH of the lysedcell composition to 10.5. Next, a salt (solid NaCl, in an amount of 2%,by weight, of the lysed cell composition) was added to the lysed cellcomposition. The lysed cell composition was then heated to a temperatureof 95° C. and held at that temperature level for 3 hours while agitatingthe lysed cell composition. A second base (i.e., a 25% solution of NaOH)was then added to the lysed cell composition until the pH was 8.07. Thelysed cell composition was then centrifuged at 5,100 rpm for 5 minutesto separate the lysed cell composition and yield a lipid layer and anemulsion layer wherein the emulsion layer was larger than the lipidlayer. In order to determine if raising the pH increased the yield ofthe lipid layer, additional second base was added to the separated lysedcell composition until the pH was 9.11 and the lysed cell compositionwas again centrifuged at 5,100 rpm for 5 minutes. This resulted in asimilar yield of lipid layer. Additional second base was added again tothe separated lysed cell composition until the pH was 10.09 and thelysed cell composition was again centrifuged at 5,100 rpm for 5 minutes.Again, the resulted in a similar yield of lipid layer.

Example 8

A cell broth containing microbial cells (Crypthecodinium cohnii) wasused in a decreased biotin trial fermentor. 20,000 kg of washed,concentrated, and pasteurized broth was harvested. This was pulled outat the startup of pasteurization. It was held for approximately 1 daybefore being transferred and homogenized. The material was homogenizedat 813 bar/one pass and collected back into a treatment tank. Throughmicroscopic inspection, it was estimated that approximately 80% of thecells were lysed.

The broth was heated to about 40° C. before treatment began. The pH wasadjusted to 10.5 and 2% NaCl was added and heated to 66° C. At thispoint a significant oil layer had already formed and the pH had droppedto 9.5 after 1-2 hours. The broth was held at 66° C. overnight.

The next day, the broth was centrifuged on the Westfalia RSE-110 with a155 mm ring dam installed. The viscosity was about 180 cP at 40° C. Thecentrifuge was fed at 48 kg/min, with 5-10 psi backpressure on the lightphase and 30 psi backpressure on the heavy phase. The feed temperaturewas maintained at 70° C. No oil was present in the waste phase and onlya few drops were visible after isopropyl alcohol was added.

Table 1 shows the results of analyses performed on the crude oilobtained from this procedure.

TABLE 1 Specifications of crude oil obtained using process of Example 8.% Oil 87.79 DHA (mg/g) 531.02 % DHA 60.49 PV 1.95 (0.6*) AV 15 % FFA0.18 Phosphorus (ppm) 8.65 Copper (ppm) 0.22 Iron (ppm) 0.7 Lead (ppm)0.63 *PV of the centrifuged oil.

Of the 20,000 kg of broth provided, 10.5% by weight (2,100 kg) wasbiomass. Of the biomass, 50% by weight was oil (1,050 kg). Of the oil,58.9% by weight was DHA (618 kg). After running the process describedabove, there was 592.5 kg of material in the lipid layer, of which about87.8% by weight (520.2 kg) was oil. Thus, the extraction yield of oilfrom the biomass was 49.5%. Of that oil, 60.5% by weight (314.6 kg) wasDHA, thereby resulting in an extraction yield of 51% by weight DHA fromthe biomass.

Example 9

A cell broth (about 500 g) that was washed, concentrated, andpasteurized containing microbial cells (Schizochytrium) was provided.The broth was chemically treated with a base (i.e., a 25% solution ofNaOH) without a prior cell lysis step. The addition of the base raisedthe pH of the broth from 5.8 to 11.2. The addition of the base and therise in the pH lysed the cells to form a lysed cell composition. Next, asalt (solid Na₂SO₄, in an amount of 5%, by weight, of the lysed cellcomposition) was added to the lysed cell composition. The lysed cellcomposition was then heated to a temperature in a range of 90° C. to 95°C. and held at that temperature level for 90 minutes and the pH of thelysed cell composition dropped to 9.7. After the 90 minutes there was anabout 2.5 ml of oil layer per 45 ml of fermentation broth and there wasno moisture loss. After 3 hours, the pH had dropped to 9.2. The solutionwas then centrifuged at about 5,100 rpm for 5 minutes to separate thelysed cell composition and yield a lipid layer. The extraction yield ofthe lipid was 81% by weight. The anisidine value (AV) of the crude oilwas 20.1. The cell breakage yield was in a range of 92% to 98% byweight.

Example 10

A cell broth (about 500 g) that was washed, concentrated, andpasteurized containing microbial cells (Schizochytrium) was provided.The broth was chemically treated with a base (i.e., a 25% solution ofNaOH) without a prior cell lysis step. The addition of the base raisedthe pH of the broth from 4.8 to 11. The addition of the base and therise in the pH lysed the cells to form a lysed cell composition. Next, asalt (solid NaCl, in an amount of 2%, by weight, of the lysed cellcomposition) was added to the lysed cell composition. The lysed cellcomposition was then heated to a temperature in a range of 90° C. to 95°C. and held at that temperature level for 3.5 hours and the pH of thelysed cell composition dropped to 8.7 and there was no moisture loss.The solution was then centrifuged at about 5,100 rpm for 5 minutes toseparate the lysed cell composition and yield a lipid layer. Theextraction yield of the lipid after 3.5 hours was 92% by weight.

A portion of the lysed cell composition was held for 6 hours and the pHof the lysed cell composition dropped to 8.6. The solution was thencentrifuged at about 5,100 rpm for 5 minutes to separate the lysed cellcomposition and yield a lipid layer. The extraction yield of the lipidafter 6 hours was 89% by weight. The anisidine value (AV) of the crudeoil was 14.4. The cell breakage yield was 95% by weight.

Example 11

A cell broth (about 500 g) that was washed, concentrated, andpasteurized containing microbial cells (Schizochytrium) was provided.The broth was chemically treated with a base (i.e., a 50% solution ofNaOH) without a prior cell lysis step. The addition of the base raisedthe pH of the broth from 5.8 to 11.2. The addition of the base and therise in the pH lysed the cells to form a lysed cell composition. Thelysed cell composition was then heated to 70° C. under vacuum to reducethe water content from 88.7% to 85.5%. During evaporation, the pHdropped to 10.36. The solution was then centrifuged at about 5,100 rpmfor 5 minutes to separate the lysed cell composition and yield a lipidlayer. The extraction yield of the lipid was 83.9% by weight. Theanisidine value (AV) of the crude oil was 10.5. The cell breakage yieldwas 93.17% by weight.

The process was repeated except the lysed cell composition was heated to70° C. under vacuum to reduce the water content from 88.7% to 79.2%. Theextraction yield of the lipid was 87.5% by weight when the water contentwas reduced to 79.2% and the cell breakage yield was 92.3% by weight.The process was also repeated to reduce the water content from 88.7% to80.8%. The extraction yield of the lipid was 90% by weight when thewater content was reduced to 80.8% and the cell breakage yield was 95.9%by weight,

Example 12

A cell broth (about 500 g) that was washed, concentrated, andpasteurized containing microbial cells (Schizochytrium) was provided.The broth was chemically treated with a base (i.e., a 50% solution ofNaOH) without a prior cell lysis step. The addition of the base raisedthe pH of the broth from 5.6 to 11.1. The addition of the base and therise in the pH lysed the cells to form a lysed cell composition. Thelysed cell composition was then heated to 90° C. in a closed system for40 minutes. After the 40 minutes, there was an about 1 ml layer of oil(lipid) per 40 ml of fermentation broth. The solution was thencentrifuged at about 5,100 rpm for 5 minutes to separate the lysed cellcomposition and yield a lipid layer. The extraction yield of the lipidwas 85.1% by weight. The anisidine value (AV) of the crude oil was 16.3.The cell breakage yield was 97.6% by weight.

Example 13

A cell broth (about 500 g) that was washed, concentrated, andpasteurized containing microbial cells (Schizochytrium) was provided.The broth was chemically treated with a base (i.e., a 50% solution ofNaOH) without a prior cell lysis step. The addition of the base raisedthe pH of the broth from 4.9 to 11.2. The addition of the base and therise in the pH lysed the cells to form a lysed cell composition. Thelysed cell composition was then mixed at room temperature for 4 hours.The solution was then centrifuged at about 5,100 rpm for 5 minutes toseparate the lysed cell composition and yield a small lipid layer.

A portion of the lysed cell composition was mixed at room temperaturefor about 96 hours. The solution was then centrifuged at about 5,100 rpmfor 5 minutes to separate the lysed cell composition and yield a largerlipid layer. The extraction yield of lipid was 61.4% by weight. Theanisidine value (AV) of the crude oil was 22.6.

Example 14

A cell broth (about 500 g) that was washed, concentrated, andpasteurized containing microbial cells (ATCC Accession No. PTA-9695) wasprovided. The broth was chemically treated with a base (i.e., a 50%solution of NaOH) without a prior cell lysis step. The addition of thebase raised the pH of the broth from 5.6 to 11.1. The addition of thebase and the rise in the pH lysed the cells to form a lysed cellcomposition. The lysed cell composition was then heated in a range of70° C. to 75° C. for 3 hours. The solution was then centrifuged at about5,100 rpm for 5 minutes to separate the lysed cell composition and yielda lipid layer. The extraction yield of the lipid was 84.4% by weight.

A portion of the lysed cell composition was heated for a total of 5hours. The solution was then centrifuged at about 5,100 rpm for 5minutes to separate the lysed cell composition and yield a similar lipidlayer. The extraction yield of lipid was 87.3% by weight. The cellbreakage yield was 89.1% by weight.

Example 15

A cell broth (about 500 g) that was washed, concentrated, andpasteurized containing microbial cells (Schizochytrium) was provided.The broth was chemically treated with a base (i.e., a 50% solution ofNaOH) without a prior cell lysis step. The addition of the base raisedthe pH of the broth from 7.3 to 11. The addition of the base and therise in the pH lysed the cells to form a lysed cell composition. Next, asalt (solid Na₂SO₄, in an amount of 5%, by weight, of the lysed cellcomposition) was added to the lysed cell composition. The lysed cellcomposition was then heated to a temperature of 90° C. and held at thattemperature level for 2 hours. While maintaining a temperature of 90° C.for additional 2 to 4 hours, the vessel containing the lysed cellcomposition was opened to allow evaporation of water. The solution wasthen centrifuged at about 5,100 rpm for 5 minutes to separate the lysedcell composition and yield a lipid layer. Extraction yield of the lipidwas greater than 70% by weight. The anisidine value (AV) of the crudeoil was 11.6.

Example 16

A cell broth (9,925 kg) containing microbial cells (ATCC Accession No.PTA-9695) was provided. The cell broth was diluted with water in a 1:1ratio by weight to form a diluted broth of 20,000 kg. The solid contentof the broth prior to dilution was 16.13% by weight and after dilutionwas 8.25% by weight. The diluted broth was mixed and centrifuged with adesludging centrifuge at 6,400 rpm to remove extracellular water-solubleor water-dispersible compounds. The concentrate (10,250 kg) from thecentrifuge was collected and had a solids content of 10.5% by weight.The collected concentrate was heated to 62° C. to 64° C. to pasteurizethe concentrate. Enzymes (i.e., Alcalase 2.4 L FG 0.5%) were added tothe pasteurized concentrate to lyse the cells and form an emulsifiedlysed cell composition. The emulsified lysed cell composition wastreated with a base (i.e., a 25% solution of NaOH) to adjust the pH ofthe lysed cell composition to 11. Next, a salt (solid Na₂SO₄, in anamount of 5%, by weight, of the lysed cell composition) was added to thelysed cell composition. The lysed cell composition was then heated to atemperature of 95° C. and held at that temperature level for 10 hours to12 hours while agitating the lysed cell composition. After agitation,the pH of the lysed cell composition was 8.6 and there was a very smallemulsion layer. The agitation tank was allowed to cool to 60° C. and thepH of the lysed cell composition increased to 9.6 while cooling. The pHof the lysed cell composition was lowered to 8.2 by adding phosphoricacid. The addition of the phosphoric acid did not harm the separation ofthe lipid layer and the very small emulsion layer. The lysed cellcomposition was then centrifuged at 5,100 rpm at a feed rate of 48kg/min for 5 minutes at 60° C. to 63° C. to separate the lysed cellcomposition and yield a lipid layer having a moisture content of 1.7% to2.3% by weight.

Example 17

A cell broth (500 g) that was washed, concentrated, and pasteurizedcontaining microbial cells (Crypthecodinium cohnii) was provided. Thebroth was homogenized at a pressure of 8,000 to 12,000 psi (2 passes) toform a lysed cell composition. The lysed cell composition was treatedwith a base (i.e., a 12.5% solution of NaOH) until the lysed cellcomposition reached a pH of 7.8 to 8.2. A salt (solid Na₂SO₄, in anamount of 5% by weight of the lysed cell composition) was added to thelysed cell composition. The lysed cell composition was then heated to atemperature of 60° C. and held at that temperature. The pH of the lysedcell composition was maintained at the 7.8 to 8.2 level by the additionof base (i.e., a 12.5% solution of NaOH) for 10 to 15 hours in a closedsystem with little to no moisture loss. The lysed cell composition wasthen centrifuged at about 5,100 rpm for 5 minutes to separate the lysedcell composition and yield an oil layer. This resulted in an oil layerof about 2 ml in a sample of 40 ml. The extraction yield of the oil was73% by weight. The anisidine value (AV) of the crude oil was 13.5. Thecell breakage yield was 82% to 86% by weight.

Example 18

A pasteurized cell broth (1,000 g) containing microbial cells(Schizochytrium) was provided. Enzymes (i.e., Alcalase 2.4 L FG 0.5%)were added to the cell biomass to lyse the cells and form an emulsifiedlysed cell composition. The emulsified lysed cell composition wastreated with a base (i.e., a 12.5% solution of NaOH) to adjust the pH ofthe lysed cell composition from 7.21 to 10.52. Next, a salt (solid NaCl,in an amount of 2%, by weight, of the lysed cell composition) was addedto the lysed cell composition. The broth was then separated into 4portions with each portion being held at 4 different temperatures andtimes: 1) Trial #1 was held at 90° C. for 22 hours; 2) Trial #2 was heldat 90° C. for 2 hours and then held at 25° C. for 20 hours; 3) Trial #3was held at 60° C. for 22 hours; and 4) Trial #4 was held at 25° C. for22 hours. The individual trials were then centrifuged without further pHadjustment. For Trials #1, #2, and #3, the broth was centrifuged atapproximately 6,600 rpm (a g-force of 4,800) for 5 minutes to separatethe lysed cell composition. Because the separation for Trial #4 was notgood (<20%) at a g-force of 4,800, the g-force was increased to 15,000and the broth was spun at a g-force of 15,000 for 5 minutes. Theextraction yield of the lipid as a weight percent and the anisidinevalue (AV) are listed in the table below.

TABLE 2 Conditions and Results When Varying Temperature and Heating Timeof the Lysed Cell Composition. Treatment Time Centrifugation Oil YieldTrial # and Temp. Conditions AV (%) 1 90° C. for 22 hours pH = 6.22 58.751.4 g-force = 4,800 2 90° C. for 2 hours, pH = 8.19 109.2 82.2 25° C.for 20 hours g-force = 4,800 3 60° C. for 22 hours pH = 8.38 91.2 27.2g-force = 4,800 4 25° C. for 22 hours pH = 10.03 105.2 55.7 g-force=15,000

The anisidine values in Table 2 were higher than expected. Onedifference between previous examples and this example was that the lysedcell composition was allowed to sit for a long period of time before thelipids were extracted. It is hypothesized that the long period of timebefore extraction leads to the oxidation of the dissolved oxygen presentin the lysed cell composition. The increased oxidation then leads to anincrease in the anisidine value. The fact that the trial heated at thehighest temperature for the longest time (Trial #1) had the lowestanisidine value supports this hypothesis because the dissolved oxygencontent of a lysed cell composition generally decreases as thetemperature is increased. The increased anisidine values are thereforebelieved to be an anomaly that was a result of the delay in extractingthe lipids from the lysed cell composition. In production, there wouldbe no delay time in extracting the lipids from the lysed cellcomposition and the anisidine values would be consistent with previousresults of anisidine values of 26 or less.

Example 19

A pasteurized cell broth (1,000 g) containing microbial cells(Schizochytrium) was provided. The broth was then split into 3 portionsand diluted as follows: 1) Trial #1 was not diluted at all and served asthe control portion; 2) Trial #2 was diluted 25% with water; and 3)Trial #3 was diluted 50% with water. Enzymes (i.e., Alcalase 2.4 L FG0.5%) were added to the cell biomass to lyse the cells and form anemulsified lysed cell composition. The emulsified lysed cell compositionwas treated with a base (i.e., a 12.5% solution of NaOH) to adjust thepH of the lysed cell composition from 6.8 to 10.6. Next, a salt (solidNaCl, in an amount of 2%, by weight, of the lysed cell composition) wasadded to the lysed cell composition. The broth was then heated to 90° C.and held for 20 hours. After the hold time, the broth for each trial wasseparated into two with one half being centrifuged as is and the otherhalf having its pH adjusted to approximately 8.5 before centrifugation.Both portions were then centrifuged at approximately 8,545 rpm (ag-force of 8,000) for 5 minutes. The extraction yield of the lipid as aweight percent and the anisidine value (AV) are listed in the tablebelow.

TABLE 3 Conditions and Results When Varying Dilution of the PasteurizedBroth. Centrifugation Oil Yield Trial # Diluted? Conditions AV (%) 1 Nodilution pH = 6.0 51.8 81.2 g-force = 8,000 1a No dilution pH = 8.4 44.378.1 g-force = 8,000 2 25% dilution pH = 5.5 76.1 88.9 with waterg-force = 8,000 2a 25% dilution pH = 8.4 85.3 82.1 with water g-force =8,000 3 50% dilution pH = 5.7 68.5 85.0 with water g-force = 8,000 3a50% dilution pH = 8.5 79.6 84.0 with water g-force = 8,000

The anisidine values in Table 3 were higher than expected. Onedifference between previous examples (excluding Example 18) and thisexample was that the lysed cell composition was allowed to sit for along period of time before the lipids were extracted. It is hypothesizedthat the long period of time before extraction leads to the oxidation ofthe dissolved oxygen present in the lysed cell composition. Theincreased oxidation then leads to an increase in the anisidine value.The increased anisidine values are therefore believed to be an anomalythat was a result of the delay in extracting the lipids from the lysedcell composition. In production, there would be no delay time inextracting the lipids from the lysed cell composition and the anisidinevalues would be consistent with previous results of anisidine values of26 or less.

Example 20

Cell broths obtained from various fermentation lots were treated usingthe process described in Example 2 except the timing of adding the salt(e.g., before and after homogenization) and the amount of salt werevaried. The resulting separated lipids were analyzed and the analysesare provided in Table 4.

TABLE 4 Specifications of lipids obtained using processes of the presentinvention varying the timing of salt addition and the amount of saltadded. Fermentation Lot P2137 P2137 P4167 P4167 P2137 P2137 Addition ofBefore Before After After After After NaCl* % NaCl by 2 5 2 5 5 5 weight% Lipid* 27 37 51 72 59 15 % Starting 13 12.9 19.2 19.2 13.3 7.5⁺ Solidsby weight % Solids by 19.7 21.7 19.7 20.1 20.2 8.6 weight beforeCentrifugation *Addition of NaCl was before or after homogenization; %lipid refers to the percentage of lipids in triglyceride form ⁺This runwas diluted to have a low percentage of starting solids.

The data provided in Table 4 demonstrates that adding the salt afterhomogenization results in higher % lipid values than adding the saltbefore homogenization. The data provided in Table 4 also demonstratesthat diluting the sample resulted in lower a % lipid value.

Example 21

A sample of Alcalase enzyme treated lysed cell composition obtained frommicrobial cells (Schizochytrium) was used. The sample had a pH ofapproximately 5.5. The sample was divided into 4 smaller samples and thepH of three of the samples was adjusted to approximately 7.4,approximately 10.5, and approximately 12, respectively, by adding sodiumhydroxide. The samples were diluted in a 1:1 ratio with deionized water.POBN (α-(4-Pyridyl 1-oxide)-N-tert-butylnitrone, 1.25 M; 50 μL) wasadded as a spin trap chemical to 0.5 g of each of the diluted samples.The samples were measured with a Bruker BioSpin e-scan EPR (ElectronParamagnetic Resonance) spectrometer (system number SC0274) (BrukerBioSpin, Billerica, Mass.) to measure the amount of free radicalspresent from lipid oxidation. The samples were incubated at roomtemperature (20° C.) and 50 μL of each of the POBN containing sampleswas tested at hourly intervals for four hours after adjusting the pHsusing the following spectrometer parameters: modulation frequency of 86Hz, modulation amplitude of 2.11 gauss, microwave power of 5.19 mW, timeconstant of 20.48 seconds, sweep time of 10.49 seconds, sweep width of100 gauss, and a number of scans of 8. The results of the EPRspectrometer readings are provided in FIG. 5.

The data in FIG. 5 demonstrates that initially the level of freeradicals was highest for the sample at pH 5.5 and lowest for the samplesat pH 10.5 and 12. The data also demonstrates that over the 4 hourperiod the rate of radical formation was slowest for the sample at pH10.5 and highest for the sample at pH 5.5. The data also demonstratesthat the addition of a base to the lysed cell composition inhibits lipidoxidation, and therefore leads to a low AV in the crude lipid andrefined oil.

Example 22

Oilseeds are extracted from a rapeseed plant and are then passed througha grinding mill to crack and break the outer hull of the oilseeds. Theoilseeds are then dehulled through known means, such as throughaspiration, to remove the meat (interior) of the seeds from the hull ofthe oilseeds. The dehulled oilseeds are then homogenized or expelled bypassing them through a press to grind the dehulled oilseeds into a cakein order to lyse the cells of the oilseeds. Water is added to form anemulsified lysed cell composition. The emulsified lysed cell compositionis filtered to remove any excess hull fragments from the lysed cellcomposition. The emulsified lysed cell composition is treated with abase (i.e., a 25% solution of NaOH) to adjust the pH of the lysed cellcomposition to 11. Next a salt (solid NaCl, in an amount 2% by weight ofthe lysed cell composition) is added to the lysed cell composition. Thelysed cell composition is then heated to a temperature of 90° C. andheld at that level for 6 hours to 48 hours while agitating the lysedcell composition. The lysed cell composition is then centrifuged at5,100 rpm for 5 minutes to separate the lysed cell composition and yielda lipid layer and an emulsion layer.

Example 23 Comparative Analysis of Crude Lipids Obtained by HexaneExtraction

The crude lipids obtained from a lot using the process described inExample 2 was analyzed to determine various specifications. Additionalcrude lipids were obtained using a typical hexane extraction process onthe same microbial cell utilized in Example 2. The hexane extractionprocess included spray drying a fermentation broth, adding hexane to thespray dried biomass to obtain a solution of 15% to 20% solids by weight.The solution was then homogenized to lyse the cells to form a lysed cellcomposition. The lysed cell composition was centrifuged and a layercontaining lipid and hexane was removed. The hexane was then removedfrom the lipid. The results of the analyses are provided in Table 5.

TABLE 5 Specifications of lipids obtained using processes of the presentinvention or a hexane extraction process. Fermentation Lot A B C D E F GExtraction Method Ex. 2 Hexane Hexane Hexane Hexane Hexane Hexane AV*5.9 ND ND ND 14.7 17.18 6.7 PV* 1.21 0.65 1.56 0.46 ND 0.85 0.3 % Lipid*89.61 86.94 84.31 86.75 85.53 86.05 86.54 DHA (mg/g) 537.47 508.15459.32 465.31 510.49 495.82 506.33 % DHA* 59.98 58.39 54.49 53.65 59.7157.68 58.51 *AV = Anisidine Value; PV = peroxide value; % lipid refersto the percentage of lipids in triglyceride form; % DHA refers to thepercentage of DHA in the lipid

The data provided in Table 5 demonstrates that the crude lipids obtainedby the processes of the present invention exhibit superior anisidinevalues, percentage of lipid, amount of DHA and percentage of DHAcompared to lipids prepared by typical hexane extraction processes.

Comparative Analysis of Crude Lipids Obtained by the FRIOLEX® ProcessesExample 24

The crude lipids obtained from various fermentation lots using theprocesses described in Examples 1 and 3 were analyzed to determinevarious specifications. Additional crude lipids were obtained using aFRIOLEX® process (Westfalia Separator AG, Germany), which is a processof extracting lipids with a water-soluble organic solvent as describedin U.S. Pat. No. 5,928,696 and International Pub. Nos. WO 01/76385 andWO 01/76715. The results of the analyses are provided in Table 6.

TABLE 6 Specifications of lipids obtained using processes of the presentinvention or a FRIOLEX ® process. Fermentation Lot A B B B B C C CExtraction Method Ex. 1 Ex. 1 Ex. 3 Ex. 1^(a) Ex. 1^(b) Ex. 1 Ex. 1FRIOLEX ® AV* 3.1 1.6 3.9 300 7.1 3.5 4 36 PV* 1.8 0.17 0.14 6.16 0.34 00 0.35 % Lipid* 96.27 93.67 92.55 87.20 94.42 95.14 94.31 93.92 DHA(mg/g) 452.4 458.16 455.17 414.66 471.55 416.41 416.05 415.38 ExtractionYield (%) 93.5 87 ND 96 ND 94 94 94 *AV = Anisidine Value; PV = peroxidevalue; % lipid refers to the percentage of lipids in triglyceride form^(a)The lysed cell composition was not heated. ^(b)The lysed cellcomposition was allowed to stand for 3 weeks prior to extraction.

The data provided in Table 6 demonstrates that the crude lipids obtainedby the processes of the present invention exhibit superior anisidinevalues (with the exception of the lipid obtained when the lysed cellcomposition was not heated) compared to lipids prepared by a FRIOLEX®process. The lipids prepared by a process of the present inventionexhibit anisidine values that are from 4.4% to 19.7% of the anisidinevalues of a lipid prepared using the FRIOLEX® process.

It is believed that a lipid prepared by a process of the presentinvention has increased stability. For example, as shown in Table 6, aprocess of the present invention was used to extract a lipid from alysed cell composition, wherein the lysed cell composition was allowedto stand for 3 weeks prior to the extraction process. It is believedthat the anisidine value of a lipid in a lysed cell compositionincreases with time, and thus, it would be expected that a lipidextracted from a 3 week old lysed cell composition have increasedanisidine values. However, as shown in Table 6, the lipid obtained fromthe 3 week old lysed cell composition using a process of the presentinvention had an anisidine value which was 19.7% of the anisidine valueof a lipid prepared by the FRIOLEX® process.

Example 25

The crude lipids obtained from a broth of microbial cells (ATCCAccession No. PTA-9695) using the process described in Example 16 wereanalyzed to determine various specifications. Additional crude lipidswere obtained from a broth of microbial cells (ATCC Accession No.PTA-9695) using a FRIOLEX® process (Westfalia Separator AG, Germany),which is a process of extracting lipids with a water-soluble organicsolvent (e.g., isopropyl alcohol) as described in U.S. Pat. No.5,928,696 and International Pub. Nos. WO 01/76385 and WO 01/76715. Theresults of the analyses are provided in Table 7.

TABLE 7 Crude Lipid Comparison Example 16 FRIOLEX ® % Oil 9.19 93.73 DHA(mg/g) 570.68 574.33 % DHA 62.1 61.27 % FFA 1.13 0.22 PV 0 0.74 AV 1073.9 Iron (ppm) 0.11 0 Copper (ppm) 0.67 0.3 Lead (ppm) 0.21 0Phosphorus (ppm) 5.22 7.20 Extraction yield (%) 61.4 45.3

The data provided in Table 7 demonstrates that the crude lipids obtainedby the processes of the present invention exhibit superior anisidinevalues (AV) and peroxide values (PV) compared to lipids prepared by aFRIOLEX® process. The data also demonstrates that the extraction yieldof lipid obtained by the processes of the present invention are superiorcompared to the extraction yield of lipid obtained by a FRIOLEX®process.

Example 26

Refining of Crude Lipids

Crude lipids were obtained using the processes outlined in Example 1 anda FRIOLEX® process. The crude lipids were further processed bysequentially: 1) degumming and caustic refining; 2) bleaching; 3)chilled filtering; and 4) deodorizing with antioxidants. The data forthe crude lipids, caustic refined lipids, bleached lipids, anddeodorized lipids are presented in Tables 8a and 8b. A comparison of therefined oils is presented in Table 9.

TABLE 8a Lipids obtained from the FRIOLEX ® process (Example 24) FFA DHAYield % Yield % Processing Step % PV AV (mg/g) (Lipid) (DHA) Crude Lipid0.28 0.37 33.6 413.6 N/A N/A Caustic Refined Lipid <0.1 0.29 — 416.485.7 86.3 Bleached Lipid <0.1 0.16 13.9 413.1 71.5 70.9 Filtered Lipid<0.1 0.19 13.3 424.86 70.3 76.0 Deodorized Lipid 0.06 <0.1 14.3 401.596.6 89.4 w/AOX* *AOX refers to antioxidants

TABLE 8b Lipids obtained from an extraction process using NaCl(Example 1) DHA Yield % Yield % Processing Step FFA % PV AV (mg/g)(Lipid) (DHA) Crude Lipid 1.36 0 3.5 406.7 N/A N/A Caustic Refined <0.10.27 2.3 410.8 85.9 86.7 Lipid Bleached Lipid <0.1 0.16 0.8 404.3 97.095.5 Filtered Lipid <0.1 0.37 1.0 414.1 59.3 60.7 Deodorized Lipid 0.06<0.1 1.8 379.9* 94.9 87.1 w/AOX *Note: Increased dilution with higholeic sunflower oil (HOSO) was the reason for the decrease in DHA(mg/g).

TABLE 9 Refined Oil Comparison FRIOLEX ® (Example 24) Example 1 DHA(mg/g) 401.5 379.9* % DHA 42.69 40.18 FFA % 0.06 0.06 PV <0.1 <0.1 AV14.3 1.8 Iron (ppm) 0.05 <0.02 Copper (ppm) <0.02 <0.02 Lead (ppm) <0.1<0.1 Arsenic (ppm) <0.1 <0.1 Mercury (ppm) <0.01 <0.01 % Moisture andvolatiles <0.01 <0.01 Unsaponifiables (%) 1.17 1.33 Trans-fatty acid byIR (%) <1 <1 *Note: Increased dilution with HOSO was the reason for thedecrease in DHA (mg/g).

The data provided in Table 8a, Table 8b, and Table 9 demonstrate that arefined oil prepared by a process of the present invention exhibitslower anisidine values compared to a refined oil prepared by theFRIOLEX® process.

Example 27 Sensory Profile Comparison

The refined oils obtained in Example 26 were analyzed by a panel of 8 to12 sensory analysts. The sensory analysts rated various lipidspecifications based on aroma, aromatics, and aftertaste to provide an“overall aroma intensity” for each lipid. The Universal Spectrumdescriptive analysis method was used to assess the aroma and aromaticcharacteristics of samples. This method uses an intensity scale of 0-15,where 0=none detected and 15=very high intensity, to measure the aromaand aromatic attributes of the oils. The results of the sensory data areprovided in Table 10.

TABLE 10 Sensory specifications of a lipid prepared by the FRIOLEX ®process (Example 24) and a lipid prepared by a process of the presentinvention (Example 1) Specifications Aroma Overall Fishy Marine GreenNutty Intensity Complex Complex Herbaceous Roasted Painty OtherFRIOLEX ® 3 1 1 1 0 0 0 Ex. 1 2 0 1 1 0 0 0 Aromatics Overall FishyMarine Green Nutty Intensity Complex Complex Herbaceous Roasted PaintyOther FRIOLEX ® 4 1 1 2 0 0 0 Ex. 1 3 0 1 2 0 0 0 Aftertaste FRIOLEX ®Herbal/slightly fishy Ex. 1 Herbal

The data provided in Table 10 demonstrates that a refined oil preparedby a process of the present invention exhibits superior sensory datacompared to a refined oil prepared by the FRIOLEX® process. As shownabove, the lipids provided by the present invention had an overall aromaintensity of 3 and 2, whereas, the FRIOLEX® lipids provided an overallaroma intensity of 4 and 3, respectively.

Example 28 Comparative Example

An extraction process for obtaining lipids from microorganisms withoutthe use of an organic solvent is disclosed in U.S. Pat. No. 6,750,048. Acomparison of a refined oil obtained from a crude lipid prepared by aprocess of the present invention and a refined oil obtained from a crudelipid prepared by the extraction process disclosed in U.S. Pat. No.6,750,048 is provided in Table 11.

TABLE 11 Comparative data for a lipid prepared by a process of thepresent invention (Example 1) and a lipid prepared by a processdisclosed in U.S. Pat. No. 6,750,048. Example 1 U.S. Pat. No. 6,750,048DHA (mg/g) 379.9 346 % DHA 40.18 37.3 FFA % 0.06 ND PV <0.1 0.46 AV 1.8ND Iron (ppm) <0.02 0.26 Copper (ppm) <0.02 <0.05 Lead (ppm) <0.1 <0.20Arsenic (ppm) <0.1 <0.20 Mercury (ppm) <0.01 <0.20 % Moisture andvolatiles <0.01 0.02 Unsaponifiables (%) 1.33 ND Trans-fatty acid by IR(%) <1 ND

The data provided in Table 11 demonstrates that a refined oil obtainedfrom a crude lipid prepared by a process of the present inventionexhibits superior properties compared to a refined oil obtained from acrude lipid prepared by the extraction process disclosed in U.S. Pat.No. 6,750,048.

Example 29

The isolated thraustochytrid (ATCC Accession No. PTA-9695) wascharacterized for taxonomic classification.

Samples were collected from intertidal habitats during low tide. Water,sediment, living plant material and decaying plant/animal debris wereplaced into sterile 50 ml tubes. Portions of each sample along with thewater were spread onto solid agar plates of isolation media. Isolationmedia consisted of: 500 ml of artificial seawater, 500 ml of distilledwater, 1 g of glucose, 1 g of glycerol, 13 g of agar, 1 g of glutamate,0.5 g of yeast extract, 0.5 g casein hydrolysate, 1 ml of a vitaminsolution (100 mg/L thiamine, 0.5 mg/L biotin, 0.5 mg B₁₂), 1 ml of atrace mineral solution (PII metals, containing per liter: 6.0 gFeCl₃6H₂O, 6.84 g H₃BO₃, 0.86 g MnCl₂4H₂O, 0.06 g ZnCl₂, 0.026CoCl₂6H₂O, 0.052 g NiSO₄H₂O, 0.002 g CuSO₄5H₂O and 0.005 g Na₂MoO₄2H₂O),and 500 mg each of penicillin G and streptomycin sulfate. The agarplates were incubated in the dark at 20-25° C. After 2-4 days the agarplates were examined under magnification, and colonies of cells werepicked with a sterile toothpick and restreaked onto a fresh plate ofmedia. Cells were repeatedly streaked onto fresh media untilcontaminated organisms were removed.

Colonies from agar plates were transferred to petri dishes withhalf-strength seawater and (1 ml) of a suspension of autoclaved newlyhatched brine shrimp larvae. The brine shrimp larvae became heavilyovergrown with clusters of sporangia after 2-3 days. Released zoosporeswere biflagellate at discharge, swimming actively away from the maturesporangium, wall remnants of which are clearly visible (in phasecontrast) after spore release. Sporangia measured 12.5 μm to 25 μm indiameter, and zoospores were 2.5 μm to 2.8 μm×4.5 μm to 4.8 μm in size.There were 8 to 24 spores per individual sporangium. Settled zoosporesenlarged and rapidly underwent binary divisions leading to tetrads,octads, and finally to clusters of sporangia. Tetrad formation commencedat a very early stage prior to maturity of the sporangia. Thesecharacteristics are in agreement with the genus Schizochytrium.

The isolated thraustochytrid (ATCC Accession No. PTA-9695) was furthercharacterized based on the similarity of its 18s rRNA gene to that ofknown species. Total genomic DNA from the thraustochytrid (ATCCAccession No. PTA-9695) was prepared by standard procedures (Sambrook J.and Russell D. 2001. Molecular cloning: A laboratory manual, 3rdedition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.)and used for PCR amplification of the 18s RNA gene. The PCRamplification of the 18s rRNA gene was carried out with primerspreviously described (Honda et. al., J. Eukaryot. Microbiol. 46(6)1999). The PCR conditions with chromosomal DNA template were as follows:0.2 μM dNTPs, 0.1 uM each primer, 8% DMSO, 200 ng chromosomal DNA, 2.5 UPfuUltra® II fusion HS DNA polymerase (Stratagene), and 1× PfuUltra®buffer (Stratagene) in a 50 μL total volume. The PCR Protocol includedthe following steps: (1) 95° C. for 2 minutes; (2) 95° C. for 45seconds; (3) 55° C. for 30 seconds; (4) 72° C. for 2 minutes; (5) repeatsteps 2-4 for 40 cycles; (6) 72° C. for 5 minutes; and (7) hold at 6° C.

PCR amplification yielded a distinct DNA product with the expected sizeusing chromosomal template described above. The PCR product was clonedinto the vector pJET1.2/blunt (Fermentas) according to themanufacturer's instructions, and the insert sequence was determinedusing supplied standard primers.

Table 12 shows a comparison of the 18s rRNA sequence from thethraustochytrid (ATCC Accession No. PTA-9695) to DNA sequences in theNational Center for Biotechnology Information (NCBI) electronicdatabase. Briefly, “% Identity” was determined by the scoring matrix“swgapdnamt” within the “AlignX” program of the VectorNTI program(Invitrogen), a standard for DNA alignment. The “% Coverage” was takenfrom the results of a Basic Local Alignment Search Tool (BLAST)calculation from the NCBI electronic database and is the percent of thequery length that is included in the aligned segments.

TABLE 12 Comparison of 18s rRNA Sequences % Identity % CoverageThraustochytrids Calculation #1 Calculation #2 Thraustochytriumaggregatum (p) 98 90 Thraustochutriidae sp. HU1 84 86 Thraustochutriidaesp. 8-7 84 91 Thraustochytrium multirudimentale 81 88 Thraustochutriidaesp. PW19 81 85 Schizochytrium sp. ATCC 20888 81 95 (p): indicatespartial sequence

As shown in Table 12, it was found that, in terms of % identity, the 18srRNA gene sequence from the thraustochytrid (ATCC Accession No.PTA-9695) is closely related, though not identical, to the 18s rRNA genesequence of T. aggregatum provided in Honda, D. et al., J. Euk. Micro.46(6): 637-647 (1999). The 18s rRNA sequence published forThraustochytrium aggregatum is a partial sequence, with an approximately71 DNA nucleotide gap in the middle of the sequence. In terms of percentcoverage, the 18s rRNA gene sequence of the isolate of the invention ismore closely related to Schizochytrium sp. ATCC 20888 than to T.aggregatum.

Highly conserved proteins such as actin and beta-tubulin have beenwidely used, along with 18s rRNA gene, as markers for assessingphylogenetic relationships between organisms (Baldauf, S. M. Am. Nat.154, S178 (1999)). Total genomic DNA from the thraustochytrid (ATCCAccession No. PTA-9695) was also used as a template for PCRamplification of both the actin and beta-tubulin genes. The PCRamplification was carried out with primers designed to conserved regionsfrom the actin and beta-tubulin DNA sequences from T. aggregatum.

The PCR conditions with chromosomal DNA template were as follows: 0.2 μMdNTPs, 0.1 uM each primer, 8% DMSO, 200 ng chromosomal DNA, 2.5 UHerculase® II fusion DNA polymerase (Stratagene), and 1× Herculase®buffer (Stratagene) in a 50 μL total volume. The PCR Protocol includedthe following steps: (1) 95° C. for 2 minutes; (2) 95° C. for 30seconds; (3) 55° C. for 30 seconds; (4) 72° C. for 2 minutes; (5) repeatsteps 2-4 for 40 cycles; (6) 72° C. for 5 minutes; and (7) hold at 6° C.

PCR amplification yielded distinct DNA products with the expected sizesusing chromosomal template described above. The respective PCR productswere cloned into the vector pJET1.2/blunt (Fermentas) according to themanufacturer's instructions, and the insert sequence of each weredetermined using supplied standard primers.

Table 13 shows identities for the actin amino acid sequence from thethraustochytrid (ATCC Accession No. PTA-9695) as compared to actinsequences available in the public database. Identities were determinedthrough use of the scoring matrix “blosum62mt2” within the “AlignX”program of the VectorNTI program, a standard for protein alignment.

TABLE 13 Comparison of Actin Protein Sequence % IdentitiesThraustochytrids % Identity Thraustochytriidae sp. RT49 98Schizochytrium sp. ATCC 20888 96 Thraustochytrium striatum 96Thraustochytrium aggregatum 96 Japonochytrium marinum 95Thraustochytrium aureum 95

Table 14 shows identities for the beta-tubulin amino acid sequence fromthe thraustochytrid (ATCC Accession No. PTA-9695) as compared tobeta-tubulin sequences available in the public database. Identities weredetermined through use of the scoring matrix “blosum62mt2” within the“AlignX” program of the VectorNTI program, a standard for proteinalignment.

TABLE 14 Comparison of Beta-Tubulin Protein Sequence % IdentitiesThraustochytrids % Identity Aplanochytrium kerguelense 100Aplanochytrium stocchinoi 100 Japonochytrium marinum 100 Labyrinthulasp. N8 100 Thraustochytriidae sp. RT49 100 Thraustochytrium aggregatum100 Thraustochytriidae sp. HU1 100 Thraustochytrium aureum 100Thraustochytrium kinnei 100 Thraustochytriidae sp. #32 100Thraustochytriidae sp. PW19 100 Schizochytrium aggregatum 100Schizochytrium sp. ATCC 20888 100

Based on the above characterizations, the isolated thraustochytrid (ATCCAccession No. PTA-9695) is believed to represent a new Schizochytriumspecies and is therefore also designated as Schizochytrium sp. ATCCPTA-9695.

Example 30

The isolated thraustochytrid (ATCC Accession No. PTA-9695) produced highlevels of cell growth under varying culture conditions, as describedbelow. Typical media and cultivation conditions are shown in Table 15.Also, high levels of fatty acids and DHA were observed (i.e., greaterthan 50% by weight of the dry cell weight were fatty acids and greaterthan 50% by weight of the fatty acid methyl esters was DHA).

TABLE 15 Vessel Media Ingredient concentration ranges NaCl g/L 12.50-25, 5-20, or 10-15 KCl g/L 1.0 0-5, 0.25-3, or 0.5-2 MgSO₄•7H₂O g/L5.0 0-10, 2-8, or 3-6 (NH₄)₂SO₄ g/L 0.6 0-10, 0.25-5, or 0.5-3 CaCl₂ g/L0.29 0.1-5, 0.15-3, or 0.2-1 T 154 (yeast g/L 6.0 0-20, 1-15, or 5-10extract) KH₂PO₄ g/L 1.2 0.1-10, 0.5-5, or 1-3 Post autoclave (Metals)Citric acid mg/L 3.5 0.1-100, 1-50, or 2-25 FeSO₄•7H₂O mg/L 10.300.1-100, 1-50, or 5-25 MnCl₂•4H₂O mg/L 3.10 0.1-100, 1-50, or 2-25ZnSO₄•7H₂O mg/L 3.10 0.1-100, 1-50, or 2-25 CoCl₂•6H₂O mg/L 0.040.001-1, 0.005-0.5, or 0.01-0.1 Na₂MoO₄•2H₂O mg/L 0.04 0.001-1,0.005-0.5, or 0.01-0.1 CuSO₄•5H₂O mg/L 2.07 0.1-100, 0.5-50, or 1-25NiSO₄•6H₂O mg/L 2.07 0.1-100, 0.5-50, or 1-25 Post autoclave (Vitamins)Thiamine mg/L 9.75 0.1-100, 1-50, or 5-25 Vitamin B12 mg/L 0.16 0.1-100,0.1-10, or 0.1-1 Ca½-panto- mg/L 3.33 0.1-100, 0.1-50, or 1-10 thenatePost autoclave (Carbon) Glucose g/L 30.0 5-150, 10-100, or 20-50Nitrogen Feed: NH₄OH mL/L 21.6 0-150, 10-100, or 15-50 Typicalcultivation conditions would include the following: pH about 6.5 - about8.5, about 6.5 - about 8.0, or about 7.0 - about 7.5 temperature: about17 - about 30 degrees Celsius, about 20 - about 25 degrees Celsius, orabout 22 to about 23 degrees Celsius dissolved oxygen: about 5 - about100% saturation, about 10 - about 80% saturation, or about 20 - about50% saturation glucose controlled @: about 5 - about 50 g/L, about 10 -about 40 g/L, or about 20 - about 35 g/L.

In carbon and nitrogen-fed cultures with 8200 ppm Cl⁻ at 22.5° C. with20% dissolved oxygen at pH 7.0, the isolate produced a dry cell weightof 140 g/L after 7 days of culture, with a fatty acid content of 70% byweight. Closed loop ammonia feed was used and the pH was maintained at7.0. The omega-3 productivity was 8.92 g/(L*day) under these conditions,with 4.7 g/L EPA (5% by weight of fatty acids) and 56.3 g/L DHA (57% byweight of fatty acids) in 7 days.

In carbon and nitrogen-fed cultures with 3640 ppm Cl⁻ at 22.5° C. with20% dissolved oxygen at pH 7.0, the isolate produced a dry cell weightof 82 g/L after 7 days of culture, with a fatty acid content of 58% byweight. The omega-3 productivity was 4.5 g/(L*day) under theseconditions, with 2.1 g/L EPA (4.3% by weight of fatty acids) and 28.5g/L DHA (58.7% by weight of fatty acids) in 7 days.

In carbon and nitrogen-fed cultures with 980 ppm Cl⁻ at 22.5° C. with20% dissolved oxygen at pH 7.0, the isolate produced a dry cell weightof 60 g/L after 7 days of culture, with a fatty acid content of 53% byweight. The omega-3 productivity was 2.8 g/(L*day) under theseconditions, with 1.1 g/L EPA (3.4% by weight of fatty acids) and 18.4g/L DHA (56.8% by weight of fatty acids) in 7 days.

Example 31

Oils were extracted from a biomass sample (Sample A) of the isolatedthraustochytrid (ATCC Accession No. PTA-9695). The biomass sample wasproduced in a carbon and nitrogen-fed culture with 980 ppm CY at 22.5°C. with 20% dissolved oxygen at pH 7.0. Oils were extracted from biomassSample A by the hexane extraction process to yield microbial oil SampleA1. Briefly, dried biomass was ground with hexane using stainless steeltubes and stainless steel ball bearings for approximately 2 hours. Theslurry was vacuum filtered and the filtrate was collected. The hexanewas removed using a rotary evaporator. Oils were also extracted frombiomass Sample A using the FRIOLEX® process (GEA Westfalia Separator UKLtd., Milton Keynes, England) to yield microbial oil Sample A2.Individual lipid classes were isolated from microbial oil Samples A1 andA2 using low pressure flash chromatography, and the weight percent ofeach class was determined. The fatty acid profile of each class wasdetermined using gas chromatography with flame ionization detection(GC-FID) as fatty acid methyl esters (FAME).

Flash Chromatography—

Flash chromatography was used to separate the lipid classes present inthe crude oils, and to determine the weight percent of each classpresent in the oils. The chromatography system utilized Silica Gel 60(EMD Chemical, Gibbstown, N.J.) with mobile phase composed of PetroleumEther and Ethyl Acetate at 3 mL/min. A step gradient was used toselectively elute each lipid class from the column. The mobile phasegradient started from 100% petroleum ether and finished with 50% ethylacetate (followed by a 100% methanol wash). Fractions were collected in10 mL test tubes using a Gilson FC 204 large-bed fraction collector(Gilson, Inc., Middleton, Wis.). Each tube was analyzed by thin layerchromatography (TLC) and the tubes containing individual lipid classes(as judged by single spots on TLC plate with expected retention factor(Rf)) were pooled, concentrated to dryness, and weighed. The totalfraction content was then determined gravimetrically.

TLC Analysis—

Thin layer chromatography was conducted on silica gel plates. The plateswere eluted using a solvent system consisting of petroleum ether:ethylether:acetic acid (80:20:1) and were visualized using iodine vapor. TheRf values of each spot were then compared with reported literaturevalues for each lipid class.

Fatty Acid Analysis—

The samples of biomass and isolated lipid classes were analyzed forfatty acid composition as FAMEs. Samples were weighed directly intoscrew cap test tubes, and 1 mL of C19:0 internal standard (NuCheck,Elysian, Minn.) in toluene and 2 mL of 1.5 N HCl in methanol was addedto each tube. The tubes were vortexed briefly and placed in a heatingblock for 2 hours at 100° C. The tubes were removed from the heatingblock, allowed to cool, and 1 mL of saturated NaCl in water was added.The tubes were vortexed again, centrifuged, and a portion of the top(organic) layer was placed in a GC vial and analyzed by GC-FID. FAME'swere quantified using a 3-point internal standard calibration curvegenerated using Nu-Chek-Prep GLC reference standard (Nu-Chek Prep, Inc.,Elysian, Minn.) and tentatively identified based on retention time.Fatty acids present were expressed as mg/g and % of total FAME.

Sample A1 was prepared by dissolving the crude oil in hexane andapplying to the head of the column. After fractionation of the sampleusing flash chromatography, the sterol ester fraction accounted for 1.2%by weight, the triacylglycerol (TAG) fraction accounted for 82.7% byweight, the free fatty acid (FFA) fraction accounted for 0.9% by weight,and the diacylglycerol (DAG) fraction accounted for 2.9% by weight ofthe crude oil. The total fatty acid profiles of the Sample A1 crude oiland isolated fractions are shown below in Table 16 and Table 17calculated as mg/g and % FAME, respectively.

TABLE 16 Sample A1 Fatty Acid Profiles Calculated as Milligrams per GramFAME Biomass Crude Oil Sterol Esters TAG FFA DAG Wt. % NA 38% 1.2% 82.7%0.9 2.9% Fatty Acid FAME (mg/g) FAME (mg/g) FAME (mg/g) FAME (mg/g) FAME(mg/g) FAME (mg/g) C12:0* 0.6 0.0 1.9 3.2 1.7 0.0 C14:0* 5.7 13.6 12.820.2 13.0 17.6 C14:1* 0.0 0.0 0.0 0.0 0.0 0.0 C15:0 1.3 3.4 3.1 3.1 2.12.6 C16:0* 105.5 239.5 222.2 274.3 183.3 225.1 C16:1* 0.0 0.0 0.8 0.00.8 0.0 C18:0* 6.4 16.4 43.1 16.8 9.8 14.0 C18:1 N9* 0.0 3.8 1.9 3.3 1.03.5 C18:1 N7 0.0 0.0 0.0 0.0 0.0 0.0 C18:2 N6* 0.0 0.0 0.0 0.0 0.0 0.0C20:0* 1.8 5.5 13.0 4.7 2.0 2.9 C18:3 N3* 0.0 0.0 0.0 0.0 0.0 0.0 C20:1N9* 0.0 0.0 0.0 0.0 0.0 0.0 C18:4 N3 0.0 0.0 0.0 0.0 0.6 0.0 C20:2 N6*0.0 0.0 0.0 0.0 0.0 0.0 C20:3 N6 0.0 0.0 0.0 0.0 0.0 0.0 C22:0* 0.0 0.87.3 0.8 0.0 1.2 C20:4 N7 0.0 0.0 0.8 0.0 0.0 0.0 C20:3 N3 0.0 0.0 0.00.0 0.0 0.0 C20:4N6* 1.0 3.4 0.0 2.6 2.0 1.9 C22:1 N9* 0.0 0.0 0.0 0.00.0 0.0 C20:4 N5 0.0 0.0 0.0 0.0 0.0 0.0 C20:4 N3 1.5 4.1 1.5 3.5 2.12.1 C20:5 N3* 18.2 39.5 3.5 38.4 30.6 42.8 C24:0* 0.0 0.0 6.3 0.0 0.00.0 C22:4 N9 0.0 0.0 0.0 0.0 0.0 0.0 C24:1 N9* 0.0 0.0 0.0 0.0 0.0 0.0C22:5 N6* 11.9 29.5 8.9 26.9 14.8 18.7 C22:5 N3* 1.1 4.7 0.9 3.6 3.4 2.7C22:6 N3* 253.5 569.7 107.3 556.5 352.8 451.4 Sum of all FAME's 408.6934.0 435.4 958.0 620.1 786.4

TABLE 17 Sample A1 Fatty Acid Profiles as a Percent of Total FAMEBiomass Crude Oil Sterol Esters TAG FFA DAG Fatty Acid % FAME % FAME %FAME % FAME % FAME % FAME C12:0* 0.1 0.0 0.4 0.3 0.3 0.0 C14:0* 1.4 1.52.9 2.1 2.1 2.2 C14:1* 0.0 0.0 0.0 0.0 0.0 0.0 C15:0 0.3 0.4 0.7 0.3 0.30.3 C16:0* 25.8 25.6 51.0 28.6 29.6 28.6 C16:1* 0.0 0.0 0.2 0.0 0.1 0.0C18:0* 1.6 1.8 9.9 1.8 1.6 1.8 C18:1 N9* 0.0 0.4 0.4 0.3 0.2 0.4 C18:1N7 0.0 0.0 0.0 0.0 0.0 0.0 C18:2 N6* 0.0 0.0 0.0 0.0 0.0 0.0 C20:0* 0.40.6 3.0 0.5 0.3 0.4 C18:3 N3* 0.0 0.0 0.0 0.0 0.0 0.0 C20:1 N9* 0.0 0.00.0 0.0 0.0 0.0 C18:4 N3 0.0 0.0 0.0 0.0 0.1 0.0 C20:2 N6* 0.0 0.0 0.00.0 0.0 0.0 C20:3 N6 0.0 0.0 0.0 0.0 0.0 0.0 C22:0* 0.0 0.1 1.7 0.1 0.00.1 C20:4 N7 0.0 0.0 0.2 0.0 0.0 0.0 C20:3 N3 0.0 0.0 0.0 0.0 0.0 0.0C20:4N6* 0.3 0.4 0.0 0.3 0.3 0.2 C22:1 N9* 0.0 0.0 0.0 0.0 0.0 0.0 C20:4N5 0.0 0.0 0.0 0.0 0.0 0.0 C20:4 N3 0.4 0.4 0.4 0.4 0.3 0.3 C20:5 N3*4.5 4.2 0.8 4.0 4.9 5.4 C24:0* 0.0 0.0 1.4 0.0 0.0 0.0 C22:4 N9 0.0 0.00.0 0.0 0.0 0.0 C24:1 N9* 0.0 0.0 0.0 0.0 0.0 0.0 C22:5 N6* 2.9 3.2 2.12.8 2.4 2.4 C22:5 N3* 0.3 0.5 0.2 0.4 0.5 0.3 C22:6 N3* 62.0 61.0 24.658.1 56.9 57.4 Sum of FAME % 100.0 100.0 100.0 100.0 100.0 100.0

Sample A2 was prepared by dissolving the crude oil in hexane andapplying to the head of the column. After fractionation of the sampleusing flash chromatography, the sterol ester fraction accounted for 0.8%by weight, the triacylglycerol (TAG) fraction accounted for 83.4% byweight, the free fatty acid (FFA) fraction accounted for 1.8% by weight,and the diacylglycerol (DAG) fraction accounted for 5.6% by weight ofthe crude oil. The total fatty acid profiles of the Sample A2 crude oiland isolated fractions are shown below in Table 18 and Table 19calculated as mg/g and % FAME, respectively.

TABLE 18 Sample A2 Fatty Acid Profiles Calculated as Milligrams per GramFAME Biomass Crude Oil Sterol Esters TAG FFA DAG Wt. % NA FAME NA 0.8%83.4% 1.8% 5.6% Fatty Acid (mg/g) FAME (mg/g) FAME (mg/g) FAME (mg/g)FAME (mg/g) FAME (mg/g) C12:0* 0.6 0.0 0.0 1.5 0.0 1.0 C14:0* 5.7 13.28.9 14.1 9.5 5.4 C14:1* 0.0 0.0 0.0 0.0 0.0 0.0 C15:0 1.3 3.3 2.8 3.42.1 2.2 C16:0* 105.5 233.7 183.8 246.1 159.7 137.3 C16:1* 0.0 0.0 0.00.8 0.0 0.0 C18:0* 6.4 16.6 23.6 16.9 11.3 5.6 C18:1 N9* 0.0 7.6 5.0 4.32.4 2.6 C18:1 N7 0.0 0.0 0.0 0.0 0.0 0.0 C18:2 N6* 0.0 2.2 0.7 1.6 0.85.1 C20:0* 1.8 5.2 12.1 5.5 2.6 1.1 C18:3 N3* 0.0 0.0 0.0 0.0 0.0 0.0C20:1 N9* 0.0 0.0 0.0 0.0 0.0 0.0 C18:4 N3 0.0 0.0 0.0 0.8 1.0 0.0 C20:2N6* 0.0 0.0 0.0 0.0 0.0 0.0 C20:3 N6 0.0 0.0 0.0 0.3 0.0 0.0 C22:0* 0.00.7 6.0 1.3 0.8 0.0 C20:4 N7 0.0 0.0 0.0 0.0 0.0 0.0 C20:3 N3 0.0 0.00.0 0.0 0.0 0.0 C20:4 N6* 1.0 3.0 0.0 3.1 2.3 1.2 C22:1 N9* 0.0 0.0 0.00.0 0.0 0.0 C20:4 N5 0.0 0.0 0.0 0.0 0.0 0.0 C20:4 N3 1.5 4.1 1.4 4.32.7 1.0 C20:5 N3* 18.2 38.6 2.7 38.6 39.5 45.5 C24:0* 0.0 0.0 4.7 0.60.0 0.3 C22:4 N9 0.0 0.0 0.0 0.0 0.0 0.0 C24:1 N9* 0.0 0.0 0.0 0.0 0.00.0 C22:5 N6* 11.9 28.2 8.6 29.6 18.0 14.7 C22:5 N3* 1.1 3.4 0.0 3.5 2.52.2 C22:6 N3* 253.5 566.7 102.2 575.0 475.3 447.2 Sum of all FAME's408.6 926.5 362.3 951.3 730.4 672.5

TABLE 19 Sample A2 Fatty Acid Profiles as a Percent of Total FAMEBiomass Crude Oil Sterol Esters TAG FFA DAG Fatty Acid % FAME % FAME %FAME % FAME % FAME % FAME C12:0* 0.1 0.0 0.0 0.2 0.0 0.2 C14:0* 1.4 1.42.4 1.5 1.3 0.8 C14:1* 0.0 0.0 0.0 0.0 0.0 0.0 C15:0 0.3 0.4 0.8 0.4 0.30.3 C16:0* 25.8 25.2 50.7 25.9 21.9 20.4 C16:1* 0.0 0.0 0.0 0.1 0.0 0.0C18:0* 1.6 1.8 6.5 1.8 1.5 0.8 C18:1 N9* 0.0 0.8 1.4 0.5 0.3 0.4 C18:1N7 0.0 0.0 0.0 0.0 0.0 0.0 C18:2 N6* 0.0 0.2 0.2 0.2 0.1 0.8 C20:0* 0.40.6 3.3 0.6 0.4 0.2 C18:3 N3* 0.0 0.0 0.0 0.0 0.0 0.0 C20:1 N9* 0.0 0.00.0 0.0 0.0 0.0 C18:4 N3 0.0 0.0 0.0 0.1 0.1 0.0 C20:2 N6* 0.0 0.0 0.00.0 0.0 0.0 C20:3 N6 0.0 0.0 0.0 0.0 0.0 0.0 C22:0* 0.0 0.1 1.7 0.1 0.10.0 C20:4 N7 0.0 0.0 0.0 0.0 0.0 0.0 C20:3 N3 0.0 0.0 0.0 0.0 0.0 0.0C20:4 N6* 0.3 0.3 0.0 0.3 0.3 0.2 C22:1 N9* 0.0 0.0 0.0 0.0 0.0 0.0C20:4 N5 0.0 0.0 0.0 0.0 0.0 0.0 C20:4 N3 0.4 0.4 0.4 0.4 0.4 0.2 C20:5N3* 4.5 4.2 0.7 4.1 5.4 6.8 C24:0* 0.0 0.0 1.3 0.1 0.0 0.0 C22:4 N9 0.00.0 0.0 0.0 0.0 0.0 C24:1 N9* 0.0 0.0 0.0 0.0 0.0 0.0 C22:5 N6* 2.9 3.02.4 3.1 2.5 2.2 C22:5 N3* 0.3 0.4 0.0 0.4 0.3 0.3 C22:6 N3* 62.0 61.228.2 60.4 65.1 66.5 Sum of FAME % 100.0 100.0 100.0 100.0 100.0 100.0

It is noted that Samples A1 and A2 were extracted using a typical hexaneextraction and a FRIOLEX® process, respectively. The fatty acid profilesof Tables 16-19 are expected to be the substantially the same if thesamples were extracted using the processes of the present invention.

Example 32

After oil was extracted from the fermentation broth using the Friolexprocess, as described in Example 31, the crude oil was further processedvia refining, bleaching, and deodorizing steps to obtain a final oil.The final oil was diluted with high oleic sunflower oil to obtainfinished commercial oil with a DHA content of approximately 400 mg/g.Individual lipid classes were isolated and the fatty acid profiles ofeach class was determined using gas chromatography with flame ionizationdetection (GC-FID) as fatty acid methyl esters (FAME).

Flash Chromatography—

Flash chromatography was used to separate the lipid classes present inthe final oil, and to determine the weight percent of each class presentin the oil. The chromatography system utilized Silica Gel 60 (EMDChemical, Gibbstown, N.J.) with mobile phase composed of Petroleum Etherand Ethyl Acetate at 3 mL/min. A step gradient was used to selectivelyelute each lipid class from the column. The mobile phase gradientstarted from 100% petroleum ether and finished with 50% ethyl acetate(followed by a 100% methanol wash). Fractions were collected in 10 mLtest tubes using a Gilson FC 204 large-bed fraction collector (Gilson,Inc., Middleton, Wis.). Each tube was analyzed by thin layerchromatography (TLC) and the tubes containing individual lipid classes(as judged by single spots on TLC plate with expected retention factor(Rf)) were pooled, concentrated to dryness, and weighed. The totalfraction content was then determined gravimetrically.

TLC Analysis—

Thin layer chromatography was conducted on silica gel plates. The plateswere eluted using a solvent system consisting of petroleum ether:ethylether:acetic acid (80:20:1) and were visualized using iodine vapor. TheRf values of each spot were then compared with reported literaturevalues for each lipid class.

Fatty Acid Analysis—

The final oil sample and isolated lipid classes were analyzed for fattyacid composition as FAMEs. Samples were weighed directly into screw captest tubes, and 1 mL of C19:0 internal standard (NuCheck, Elysian,Minn.) in toluene and 2 mL of 1.5 N HCl in methanol was added to eachtube. The tubes were vortexed briefly and placed in a heating block for2 hours at 100° C. The tubes were removed from the heating block,allowed to cool, and 1 mL of saturated NaCl in water was added. Thetubes were vortexed again, centrifuged, and a portion of the top(organic) layer was placed in a GC vial and analyzed by GC-FID. FAME'swere quantified using a 3-point internal standard calibration curvegenerated using Nu-Chek-Prep GLC reference standard (Nu-Chek Prep, Inc.,Elysian, Minn.) and tentatively identified based on retention time.Fatty acids present were expressed as mg/g and % of total FAME.

The sample was prepared by dissolving 250 mg of final oil in 600 μL ofhexane and applying to the head of the column. After fractionation ofthe sample using flash chromatography, the sterol ester fractionaccounted for 1.2% by weight, the triacylglyceride (TAG) fractionaccounted for 92.1% by weight, the free fatty acid (FFA) fractionaccounted for 2.1% by weight, the sterol fraction accounted for 1.1%,the diacylglyceride (DAG) fraction accounted for 2.8% by weight of thefinal oil.

The TLC analysis of the pooled fractions showed that the FFA and sterolfractions were mixed with TAG and DAG respectively. The total fatty acidprofiles of the FRIOLEX® final oil and isolated fractions are shownbelow in Table 20 and Table 21 calculated as mg/g and % FAME,respectively.

TABLE 20 Fatty Acid Profile Calculated as Milligrams per Gram of FAMEFinal Sterol Oil Esters TAG FFA Sterol DAG Wt. % NA 1.2 92.1 2.1 1.1 2.8FAME FAME FAME FAME FAME FAME Fatty Acid (mg/g) (mg/g) (mg/g) (mg/g)(mg/g) (mg/g) C12:0* 0.0 0.0 1.0 0.0 1.2 0.6 C14:0* 11.5 5.1 11.3 6.09.6 5.7 C14:1* 0.0 0.0 0.0 0.0 0.0 0.0 C15:0 2.3 0.0 2.3 1.2 2.0 1.9C16:0* 183.3 80.0 180.8 99.9 149.3 132.2 C16:1* 0.0 0.0 0.9 0.0 0.8 0.6C18:0* 19.6 17.5 19.6 7.5 16.2 6.7 C18:1 N9* 243.3 242.8 249.6 62.9190.5 84.0 C18:1 N7 1.9 1.7 2.0 0.8 1.9 0.9 C18:2 N6* 13.8 5.6 13.8 6.214.3 9.1 C20:0* 4.3 6.6 4.5 1.5 3.6 1.4 C18:3 N3* 0.0 0.0 0.3 0.0 0.00.0 C20:1 N9* 0.0 0.0 0.8 0.0 0.8 0.0 C18:4 N3 0.0 0.0 0.7 1.3 0.9 0.4C20:2 N6* 0.0 0.0 0.6 0.0 0.0 0.0 C20:3 N6 0.0 0.0 0.3 0.0 0.0 0.0C22:0* 3.3 61.0 3.2 1.1 3.0 1.2 C20:4 N7 0.0 0.0 0.0 0.0 0.0 0.0 C20:3N3 0.0 0.0 0.0 0.0 0.0 0.0 C20:4N6* 1.7 0.0 2.3 1.4 1.9 1.3 C22:1 N9*0.0 0.0 0.0 0.0 0.0 0.0 C20:4 N5 0.0 0.0 0.0 0.0 0.0 0.0 C20:4 N3 2.44.5 3.0 2.2 2.6 1.3 C20:5 N3* 28.1 3.0 27.7 38.6 25.6 43.2 C24:0* 1.464.3 1.4 0.0 2.0 1.0 C22:4 N9 0.0 0.0 0.0 0.0 0.0 0.0 C24:1 N9* 0.0 0.00.0 0.0 0.0 0.0 C22:5 N6* 20.0 7.6 21.0 10.1 17.2 14.4 C22:5 N3* 2.8 0.03.1 3.7 3.4 2.9 C22:6 N3* 407.1 72.5 417.4 443.6 350.5 428.5 Sum of all936.1 572.1 967.6 688.0 797.3 737.3 FAME's

TABLE 21 Fatty Acid Profiles as a Percent of Total FAME Final Sterol OilEsters TAG FFA Sterol DAG % % % % % % Fatty Acid FAME FAME FAME FAMEFAME FAME C12:0* 0.0 0.0 0.1 0.0 0.2 0.1 C14:0* 1.2 0.9 1.2 0.9 1.2 0.8C14:1* 0.0 0.0 0.0 0.0 0.0 0.0 C15:0 0.2 0.0 0.2 0.2 0.2 0.3 C16:0* 19.614.0 18.7 14.5 18.7 17.9 C16:1* 0.0 0.0 0.1 0.0 0.1 0.1 C18:0* 2.1 3.12.0 1.1 2.0 0.9 C18:1 N9* 26.0 42.4 25.8 9.1 23.9 11.4 C18:1 N7 0.2 0.30.2 0.1 0.2 0.1 C18:2 N6* 1.5 1.0 1.4 0.9 1.8 1.2 C20:0* 0.5 1.1 0.5 0.20.5 0.2 C18:3 N3* 0.0 0.0 0.0 0.0 0.0 0.0 C20:1 N9* 0.0 0.0 0.1 0.0 0.10.0 C18:4 N3 0.0 0.0 0.1 0.2 0.1 0.1 C20:2 N6* 0.0 0.0 0.1 0.0 0.0 0.0C20:3 N6 0.0 0.0 0.0 0.0 0.0 0.0 C22:0* 0.4 10.7 0.3 0.2 0.4 0.2 C20:4N7 0.0 0.0 0.0 0.0 0.0 0.0 C20:3 N3 0.0 0.0 0.0 0.0 0.0 0.0 C20:4N6* 0.20.0 0.2 0.2 0.2 0.2 C22:1 N9* 0.0 0.0 0.0 0.0 0.0 0.0 C20:4 N5 0.0 0.00.0 0.0 0.0 0.0 C20:4 N3 0.3 0.8 0.3 0.3 0.3 0.2 C20:5 N3* 3.0 0.5 2.95.6 3.2 5.9 C24:0* 0.2 11.2 0.1 0.0 0.2 0.1 C22:4 N9 0.0 0.0 0.0 0.0 0.00.0 C24:1 N9* 0.0 0.0 0.0 0.0 0.0 0.0 C22:5 N6* 2.1 1.3 2.2 1.5 2.2 1.9C22:5 N3* 0.3 0.0 0.3 0.5 0.4 0.4 C22:6 N3* 43.6 12.7 43.1 64.5 44.058.1 Sum of 100 100 100 100 100 100 FAME %

It is noted that fatty acid profiles of Tables 20 and 21 were obtainedfrom samples extracted using a FRIOLEX® process. The fatty acid profilesof Tables 20 and 21 are expected to be the substantially the same if thesamples were extracted using the processes of the present invention.

Example 33

A two-day old inoculum flask of the isolated thraustochytrid (ATCCAccession No. PTA-9695) was prepared in a carbon and nitrogen-fedculture with 980 ppm Cl⁻ (thraustochytrid media).

Mutagenesis was carried out according to following procedure:

A sterile T=2 day old flask, approximately 50 ml, was poured into asterile 40 ml glass homogenizer. The culture received 50 plunges in thehomogenizer. The culture was pipeted out and filtered through a sterile50 micron mesh filter, which was placed in a 50 ml sterile tube (themesh was used as a means of retaining the larger clumps of colonieswhile letting the smaller clusters and single cells pass through the 50micron mesh.). The entire concentrated macerate was collected in asterile 50 ml tube. The macerated culture was vortexed and dilutions atlevels up to 1:100 fold were made in tubes containing thraustochytridmedia. The diluted macerate samples were vortexed prior to adding 200 μlof inoculum to a thraustochytrid media agar petri dish, 100×15 mm,containing 4-5 glass beads (3 mm glass beads). Each plate was gentlyagitated in an effort to have the beads spread the inoculum evenlyaround the plate. Beads were dumped off of plates and plates were leftto sit with covers on for approximately 5 minutes to dry. Lights in boththe sterile hood and adjoining areas were turned off as the procedurewas performed in dim light. There was minimal light available to be ableto run the procedure but only indirect and dim.

Five replicate plates were placed on the floor of the XL crosslinker(Spectronics Corporation, New York) with the lids off while the sampleswere irradiated. The crosslinker delivered power in terms of microjoulesand a level was sought that achieved a 90%-95% Kill. Five replicatecontrol plates were inoculated with un-mutagenized cells using the sameprotocol. These cell counts were used to calculate the % Kill. Once theirradiation was finished the plates were taken out, the lids werereplaced, and the plates were wrapped in parafilm followed by a wrap inaluminum foil. It was imperative that the plates grew for the first weekin the dark so that they were not able to repair the damaged genes.

Plates were placed in a 22.5° C. room for about 10 days prior tocounting the colonies. When final counts were made, individual colonieswere picked with a sterile inoculating loop and re-streaked on newthraustochytrid media plates. Each colony was plated on an individualplate. As plates grew dense a sample was taken, using a inoculatingloop, and inoculated into a sterile 250 ml shake flask containing 50 mlof thraustochytrid media. This flask was placed on a shaker at 200 rpmin a 22.5° C. room. On T=7 days the shake flask culture was harvestedinto a 50 ml sterile tube. The pH was taken and the sample was spun downto collect the biomass pellet. Each sample was rinsed and re-suspendedin a 50:50 mixture of isopropyl alcohol and distilled water prior tobeing re-spun. The collected pellet was freeze dried, weighed, and aFAME analysis was performed. The data in Tables 22-28 represents mutantsproduced with the above process.

TABLE 22 Mutants of Thraustochytrid Strain ATCC Accession No. PTA-9695control Fatty Acids ATCC PTA-9695 Mutant 1 Mutant 2 Mutant 3 Mutant 4Mutant 5 Mutant 8 Mutant 9 Mutant 10 % 08:0 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 % 09:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00% 10:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 11:0 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 % 11:1 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 % 12:0 0.10 0.10 0.08 0.08 0.13 0.07 0.11 0.08 0.08 %12:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 13:0 0.11 0.11 0.170.13 0.12 0.18 0.11 0.15 0.14 % 13:1 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 % 14:0 1.79 1.85 1.49 1.37 2.36 1.29 1.85 1.72 1.57 % 14:10.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 15:1 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 % 16:0 30.98 28.75 29.96 29.97 30.33 29.8630.97 30.11 29.20 % 16:1 0.27 0.20 0.31 0.14 0.25 0.27 0.16 0.27 0.24 %16:2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 16:3 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 % 17:0 0.12 0.15 0.13 0.17 0.27 0.12 0.160.13 0.13 % 18:0 1.29 1.22 1.38 1.47 1.22 1.57 1.25 1.34 1.34 % 18:1 n-90.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 18:1 n-7 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 % 18:2 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 % 18:3 n-6 0.00 0.03 0.00 0.00 0.07 0.00 0.03 0.00 0.00 % 18:3n-3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 18:4 n-3 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 % 20:0 0.39 0.36 0.42 0.45 0.34 0.460.37 0.40 0.40 % 20:1 n-9 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 %20:2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 20:3 n-9 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 % 20:3 n-6 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 % 20:3 n-3 0.37 0.38 0.32 0.42 0.44 0.32 0.41 0.330.36 % 20:4 ARA 0.55 0.55 0.94 0.57 0.80 0.89 0.60 0.73 0.75 % 20:5 n-3EPA 2.62 2.94 3.01 2.40 3.64 2.83 2.54 2.81 2.81 % 22:0 0.08 0.08 0.090.09 0.07 0.10 0.07 0.09 0.09 % 22:1 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 % 22:2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 22:30.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 22:4 n-6 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 % 22:5 n-6 3.19 3.19 2.94 3.43 3.35 2.873.34 3.01 3.15 % 22:5 n-3 0.18 0.18 0.21 0.23 0.20 0.18 0.20 0.17 0.18 %22:6 n-3 DHA 56.88 58.63 57.56 57.85 54.87 57.98 56.62 57.53 58.52 %24:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 24:1 0.00 0.08 0.000.00 0.00 0.00 0.00 0.09 0.00 % Fat 46.83 46.10 31.23 47.39 49.78 30.6254.71 37.72 37.87 % Unknown 0.85 0.46 0.35 0.51 0.51 0.36 0.50 0.38 0.39

TABLE 23 Mutants of Thraustochytrid Strain ATCC Accession No. PTA-9695control ATCC Fatty Acids PTA-9695 Mutant 11 Mutant 13 Mutant 14 Mutant15 Mutant 16 Mutant 20 Mutant 21 Mutant 22 % 08:0 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 % 09:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 % 10:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 11:0 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 11:1 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 % 12:0 0.10 0.10 0.08 0.09 0.11 0.11 0.09 0.09 0.10% 12:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 13:0 0.11 0.150.16 0.14 0.13 0.12 0.17 0.16 0.13 % 13:1 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 % 14:0 1.79 1.89 1.43 1.75 1.83 1.98 1.76 1.77 1.81 %14:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 15:1 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 % 16:0 30.98 31.08 30.27 29.92 31.79 30.1828.84 30.05 30.81 % 16:1 0.27 0.32 0.26 0.28 0.21 0.24 0.23 0.23 0.33 %16:2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 16:3 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 % 17:0 0.12 0.24 0.15 0.13 0.15 0.12 0.140.16 0.14 % 18:0 1.29 1.36 1.44 1.31 1.36 1.21 1.28 1.34 1.33 % 18:1 n-90.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 18:1 n-7 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 % 18:2 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 % 18:3 n-6 0.00 0.05 0.00 0.00 0.00 0.03 0.00 0.00 0.00 % 18:3n-3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 18:4 n-3 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 % 20:0 0.39 0.38 0.42 0.39 0.40 0.370.37 0.38 0.38 % 20:1 n-9 0.00 0.00 0.06 0.00 0.00 0.00 0.00 0.00 0.00 %20:2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 20:3 n-9 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 % 20:3 n-6 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 % 20:3 n-3 0.37 0.43 0.36 0.33 0.36 0.37 0.33 0.350.34 % 20:4 ARA 0.55 0.79 0.72 0.80 0.64 0.62 0.83 0.73 0.69 % 20:5 n-3EPA 2.62 3.17 2.72 2.97 2.52 2.66 3.03 2.90 2.87 % 22:0 0.08 0.08 0.090.08 0.08 0.08 0.08 0.08 0.08 % 22:1 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 % 22:2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 22:30.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 22:4 n-6 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 % 22:5 n-6 3.19 3.25 3.06 2.97 3.07 3.162.98 3.01 3.02 % 22:5 n-3 0.18 0.20 0.19 0.17 0.19 0.16 0.17 0.18 0.18 %22:6 n-3 DHA 56.88 55.17 57.52 57.63 56.02 57.38 58.58 57.45 56.65 %24:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 24:1 0.00 0.00 0.000.00 0.00 0.07 0.00 0.00 0.08 % Fat 46.83 46.19 37.00 38.41 48.46 47.3237.71 40.23 43.55 % Unknown 0.85 0.47 0.39 0.36 0.47 0.44 0.37 0.39 0.38

TABLE 24 Mutants of Thraustochytrid Strain ATCC Accession No. PTA-9695control ATCC Fatty Acids PTA-9695 Mutant 24 Mutant 26 Mutant 27 Mutant29 Mutant 30 Mutant 33 Mutant 34 Mutant 35 % 08:0 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 % 09:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 % 10:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 11:0 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 11:1 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 % 12:0 0.10 0.11 0.09 0.09 0.08 0.08 0.10 0.11 0.09% 12:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 13:0 0.11 0.120.13 0.14 0.16 0.14 0.12 0.12 0.10 % 13:1 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 % 14:0 1.79 1.98 1.71 1.69 1.63 1.66 1.93 2.01 1.59 %14:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 15:1 0.00 0.00 0.000.00 0.00 0.00 0.70 0.54 0.39 % 16:0 30.98 30.61 30.32 30.21 29.70 29.5030.26 32.28 30.78 % 16:1 0.27 0.19 0.22 0.22 0.26 0.26 0.29 0.26 0.16 %16:2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 16:3 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 % 17:0 0.12 0.15 0.18 0.16 0.13 0.13 0.260.16 0.12 % 18:0 1.29 1.24 1.31 1.31 1.32 1.30 1.32 1.37 1.34 % 18:1 n-90.00 0.00 0.00 0.00 0.00 0.00 0.10 0.11 0.09 % 18:1 n-7 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 % 18:2 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 % 18:3 n-6 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 18:3n-3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 18:4 n-3 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 % 20:0 0.39 0.37 0.39 0.40 0.40 0.390.37 0.40 0.40 % 20:1 n-9 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.13 0.14 %20:2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 20:3 n-9 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 % 20:3 n-6 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 % 20:3 n-3 0.37 0.38 0.37 0.35 0.35 0.35 0.00 0.000.00 % 20:4 ARA 0.55 0.61 0.59 0.69 0.68 0.32 0.34 0.24 0.28 % 20:5 n-3EPA 2.62 2.62 2.70 2.85 2.90 2.91 3.28 2.51 2.59 % 22:0 0.08 0.08 0.080.08 0.09 0.08 0.08 0.08 0.08 % 22:1 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 % 22:2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 22:30.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 22:4 n-6 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 % 22:5 n-6 3.19 3.10 3.11 3.05 3.10 3.113.43 3.26 3.56 % 22:5 n-3 0.18 0.16 0.18 0.19 0.18 0.18 0.18 0.15 0.24 %22:6 n-3 DHA 56.88 57.03 57.46 57.46 57.96 58.52 55.92 54.96 56.73 %24:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 24:1 0.00 0.08 0.000.00 0.00 0.00 0.07 0.07 0.07 % Fat 46.83 47.80 43.50 38.86 38.60 38.1646.95 46.43 51.55 % Unknown 0.85 0.45 0.42 0.39 0.37 0.82 1.25 1.23 1.25

TABLE 25 Mutants of Thraustochytrid Strain ATCC Accession No. PTA-9695control ATCC Fatty Acids PTA-9695 Mutant 36 Mutant 37 Mutant 38 Mutant39 Mutant 40 Mutant 42 Mutant 43 Mutant 44 % 08:0 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 % 09:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 % 10:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 11:0 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 11:1 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 % 12:0 0.10 0.00 0.11 0.00 0.11 0.09 0.08 0.12 0.09% 12:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 13:0 0.11 0.440.09 0.24 0.12 0.11 0.12 0.08 0.15 % 13:1 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 % 14:0 1.79 1.25 1.99 1.48 1.96 1.76 1.43 2.17 1.75 %14:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 15:1 0.00 2.12 0.480.71 0.54 0.55 0.36 0.62 0.50 % 16:0 30.98 26.95 28.04 32.28 30.84 30.2525.77 43.37 30.18 % 16:1 0.27 0.00 0.26 0.23 0.22 0.21 0.10 1.05 0.22 %16:2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 16:3 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 % 17:0 0.12 0.95 0.13 0.28 0.16 0.16 0.100.26 0.13 % 18:0 1.29 1.58 1.11 1.79 1.30 1.29 1.25 2.21 1.34 % 18:1 n-90.00 0.37 0.08 0.25 0.09 0.09 0.12 0.09 0.10 % 18:1 n-7 0.00 0.00 0.000.00 0.00 0.00 0.00 0.05 0.00 % 18:2 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 % 18:3 n-6 0.00 0.00 0.06 0.00 0.00 0.00 0.00 0.00 0.00 % 18:3n-3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 18:4 n-3 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 % 20:0 0.39 0.34 0.31 0.43 0.38 0.390.36 0.61 0.40 % 20:1 n-9 0.00 0.00 0.00 0.43 0.00 0.14 0.15 0.15 0.49 %20:2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 20:3 n-9 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 % 20:3 n-6 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 % 20:3 n-3 0.37 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 % 20:4 ARA 0.55 0.41 0.31 0.24 0.27 0.24 0.30 0.35 0.23 % 20:5 n-3EPA 2.62 5.36 2.77 4.00 2.72 2.80 3.21 3.47 2.80 % 22:0 0.08 0.00 0.070.14 0.07 0.08 0.07 0.14 0.08 % 22:1 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 % 22:2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 22:30.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 22:4 n-6 0.00 0.00 0.060.00 0.00 0.00 0.00 0.00 0.00 % 22:5 n-6 3.19 2.40 3.94 2.57 3.48 3.293.89 2.37 3.33 % 22:5 n-3 0.18 0.00 0.19 0.00 0.17 0.17 0.30 0.33 0.17 %22:6 n-3 DHA 56.88 57.52 58.57 54.20 56.24 57.09 60.99 41.61 56.76 %24:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 24:1 0.00 0.00 0.080.00 0.08 0.09 0.08 0.06 0.09 % Fat 46.83 12.73 54.86 18.08 45.74 42.5942.48 56.44 41.20 % Unknown 0.85 0.29 1.36 0.73 1.28 1.20 1.31 0.90 1.20

TABLE 26 Mutants of Thraustochytrid Strain ATCC Accession No. PTA-9695control ATCC PTA- Fatty Acids 9695 Mutant 45 Mutant 46 Mutant 47 Mutant48 Mutant 49 Mutant 50 Mutant 51 Mutant 52 % 08:0 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 % 09:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 % 10:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 11:0 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 11:1 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 % 12:0 0.10 0.10 0.13 0.11 0.07 0.09 0.09 0.09 0.11% 12:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 13:0 0.11 0.110.10 0.09 0.13 0.09 0.13 0.10 0.09 % 13:1 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 % 14:0 1.79 1.79 2.07 1.86 1.52 1.62 1.78 1.78 1.85 %14:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 15:1 0.00 0.41 0.760.57 0.46 0.48 0.55 0.53 0.53 % 16:0 30.98 28.79 24.90 30.07 29.07 31.2130.46 30.79 32.53 % 16:1 0.27 0.19 0.24 0.18 0.17 0.17 0.18 0.21 0.22 %16:2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 16:3 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 % 17:0 0.12 0.11 0.24 0.16 0.12 0.14 0.170.18 0.15 % 18:0 1.29 1.24 1.07 1.28 1.41 1.43 1.36 1.48 1.35 % 18:1 n-90.00 0.08 0.07 0.09 0.09 0.08 0.10 0.09 0.06 % 18:1 n-7 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 % 18:2 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 % 18:3 n-6 0.00 0.00 0.12 0.05 0.00 0.00 0.00 0.00 0.00 % 18:3n-3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 18:4 n-3 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 % 20:0 0.39 0.36 0.29 0.37 0.42 0.420.39 0.40 0.41 % 20:1 n-9 0.00 0.15 0.13 0.11 0.24 0.13 0.19 0.16 0.19 %20:2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 20:3 n-9 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 % 20:3 n-6 0.00 0.00 0.05 0.00 0.000.00 0.00 0.00 0.00 % 20:3 n-3 0.37 0.00 0.12 0.00 0.00 0.00 0.00 0.000.00 % 20:4 ARA 0.55 0.29 0.65 0.26 0.18 0.21 0.22 0.24 0.24 % 20:5 n-32.62 3.05 4.28 2.66 2.93 2.46 2.71 2.94 2.44 EPA % 22:0 0.08 0.07 0.060.07 0.09 0.09 0.08 0.08 0.08 % 22:1 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 % 22:2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 22:30.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 22:4 n-6 0.00 0.06 0.070.05 0.00 0.00 0.00 0.00 0.00 % 22:5 n-6 3.19 3.59 4.28 3.46 3.07 3.323.17 3.18 3.24 % 22:5 n-3 0.18 0.25 0.27 0.18 0.17 0.17 0.16 0.17 0.17 %22:6 n-3 DHA 56.88 57.74 58.32 56.70 58.65 56.45 56.83 56.19 55.06 %24:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 24:1 0.00 0.07 0.150.10 0.10 0.11 0.10 0.10 0.07 % Fat 46.83 48.91 58.95 54.80 35.41 48.6044.93 43.01 51.93 % Unknown 0.85 1.55 1.63 1.57 1.09 1.35 1.31 1.28 1.19

TABLE 27 Mutants of Thraustochytrid Strain ATCC Accession No. PTA-9695control ATCC Fatty Acids PTA-9695 Mutant 53 Mutant 54 Mutant 55 Mutant56 Mutant 57 Mutant 58 Mutant 60 Mutant 61 Mutant 65 % 08:0 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 09:0 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 % 10:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 % 11:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 %11:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 12:0 0.10 0.090.08 0.12 0.08 0.08 0.08 0.08 0.10 0.08 % 12:1 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 % 13:0 0.11 0.11 0.12 0.08 0.09 0.13 0.16 0.140.09 0.14 % 13:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 %14:0 1.79 1.74 1.63 2.13 1.67 1.59 1.59 1.59 1.85 1.58 % 14:1 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 15:1 0.00 0.53 0.52 0.48 0.510.52 0.45 0.50 0.51 0.48 % 16:0 30.98 30.13 29.54 33.01 31.08 29.3730.65 29.39 31.15 30.03 % 16:1 0.27 0.21 0.23 0.26 0.26 0.14 0.25 0.220.26 0.25 % 16:2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 %16:3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 17:0 0.12 0.150.14 0.14 0.14 0.16 0.12 0.13 0.14 0.13 % 18:0 1.29 1.30 1.30 1.37 1.381.37 1.46 1.30 1.30 1.35 % 18:1 n-9 0.00 0.08 0.08 0.00 0.06 0.11 0.090.10 0.07 0.07 % 18:1 n-7 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 % 18:2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 18:3 n-60.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 18:3 n-3 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 18:4 n-3 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 % 20:0 0.39 0.38 0.39 0.40 0.42 0.38 0.430.39 0.39 0.41 % 20:1 n-9 0.00 0.19 0.16 0.13 0.19 0.20 0.17 0.14 0.130.21 % 20:2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 20:3 n-90.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 20:3 n-6 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 20:3 n-3 0.37 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 % 20:4 ARA 0.55 0.25 0.21 0.26 0.22 0.250.51 0.20 0.24 0.19 % 20:5 n-3 EPA 2.62 2.75 2.78 2.81 2.67 2.78 5.762.72 2.59 2.82 % 22:0 0.08 0.08 0.08 0.08 0.09 0.08 0.09 0.08 0.08 0.09% 22:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 22:2 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 22:3 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 % 22:4 n-6 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 0.06 0.00 % 22:5 n-6 3.19 3.47 3.20 3.25 3.19 3.43 2.62 3.303.42 3.18 % 22:5 n-3 0.18 0.18 0.18 0.17 0.17 0.20 0.59 0.17 0.17 0.17 %22:6 n-3 DHA 56.88 56.99 58.07 54.04 56.38 57.76 54.09 58.21 55.91 57.56% 24:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 24:1 0.000.09 0.09 0.07 0.10 0.09 0.11 0.10 0.07 0.08 % Fat 46.83 45.83 39.5948.81 41.92 43.97 33.96 36.97 50.40 36.21 % Unknown 0.85 1.28 1.19 1.191.29 1.35 0.77 1.24 1.48 1.17

TABLE 28 Mutants of Thraustochytrid Strain ATCC Accession No. PTA-9695control Mutant 68 Mutant 70 Mutant 72 Fatty ATCC PTA- ATCC PTA- ATCCPTA- ATCC PTA- Mutant Acids 9695 Mutant 66 Mutant 67 9696 Mutant 69 9697Mutant 71 9698 Mutant 73 74 % 08:0 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 % 09:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00% 10:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 11:0 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 11:1 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 % 12:0 0.15 0.00 0.00 0.00 0.00 0.00 0.000.13 0.00 0.00 % 12:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00% 13:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 13:1 0.000.00 0.00 0.00 0.00 0.00 0.22 0.00 0.00 0.00 % 14:0 2.42 2.29 2.07 2.092.11 2.21 2.27 2.29 1.97 2.05 % 14:1 0.00 0.00 0.00 0.00 0.00 0.00 0.190.00 0.00 0.00 % 15:1 0.55 0.47 0.48 0.47 0.47 0.44 0.46 0.40 0.50 0.47% 16:0 39.19 31.02 26.20 25.84 27.79 28.14 28.89 33.49 24.50 23.95 %16:1 0.43 0.19 0.00 0.00 0.00 0.00 0.19 0.21 0.00 0.00 % 16:2 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 16:3 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 % 17:0 0.16 0.00 0.00 0.00 0.00 0.00 0.00 0.130.00 0.00 % 18:0 1.67 1.68 1.22 1.22 1.44 1.49 1.51 2.24 1.11 1.02 %18:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 n-9 % 18:1 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 n-7 % 18:2 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 % 18:3 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 n-6 % 18:3 0.00 0.18 0.20 0.21 0.19 0.17 0.22 0.160.22 0.22 n-3 % 18:4 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00n-3 % 20:0 0.49 0.41 0.32 0.31 0.35 0.37 0.44 0.52 0.29 0.27 % 20:1 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 n-9 % 20:2 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 % 20:3 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 n-9 % 20:3 0.00 0.00 0.00 0.00 0.15 0.00 0.00 0.000.00 0.00 n-6 % 20:3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00n-3 % 20:4 0.18 0.16 0.33 0.27 0.24 0.37 0.30 0.27 0.38 0.39 ARA % 20:51.76 2.30 3.86 3.97 3.32 4.12 3.09 2.74 4.43 4.53 n-3 EPA % 22:0 0.330.46 0.35 0.44 0.48 0.38 0.43 0.12 0.35 0.34 % 22:1 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 % 22:2 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 % 22:3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00% 22:4 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 n-6 % 22:5 2.622.83 3.17 2.66 2.72 2.95 3.46 2.79 3.17 3.19 n-6 % 22:5 0.18 0.18 0.460.42 0.34 0.61 0.25 0.27 0.48 0.57 n-3 % 22:6 49.52 57.01 60.60 61.4259.74 58.03 55.62 53.06 61.83 62.23 n-3 DHA % 24:0 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 % 24:1 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 % Fat 52.70 49.32 48.51 49.49 48.80 53.65 40.38 63.4048.27 46.63 % 0.35 0.82 0.73 0.66 0.67 0.73 2.46 1.18 0.78 0.76 Unknown

Example 34

Isolation of Microorganisms

Samples were collected from intertidal habitats during low tide,including bays and estuaries along the West Coast of North America(California, Oregon, and Washington) and Hawaii. Water, sediment, livingplant material, and decaying plant/animal debris were placed intosterile 50 ml tubes. Portions of each sample along with the water werespread onto solid agar plates of isolation media. Isolation mediaconsisted of: 500 ml of artificial seawater, 500 ml of distilled water,1 g of glucose, 1 g of glycerol, 13 g of agar, 1 g of glutamate, 0.5 gof yeast extract, 0.5 g casein hydrolysate, 1 ml of a vitamin solution(100 mg/L thiamine, 0.5 mg/L biotin, 0.5 mg B₁₂), 1 ml of a tracemineral solution (PII metals, containing per liter: 6.0 g FeCl₃6H₂O,6.84 g H₃BO₃, 0.86 g MnCl₂4H₂O, 0.06 g ZnCl₂, 0.026 CoCl₂6H₂O, 0.052 gNiSO₄H₂O, 0.002 g CuSO₄5H₂O and 0.005 g Na₂MoO₄2H₂O), and 500 mg each ofpenicillin G and streptomycin sulfate. The agar plates were incubated inthe dark at 20-25° C. After 2-4 days the agar plates were examined undermagnification, and colonies of cells were picked with a steriletoothpick and restreaked onto a fresh plate of media. Cells wererepeatedly streaked onto fresh media until contaminated organisms wereremoved. Two of the isolated microorganisms were deposited under ATCCAccession Nos. PTA-10212 and PTA-10208.

Taxonomic Characteristics of the Isolated Microorganism Deposited UnderATCC Accession No. PTA-10212

Cultures of the isolated microorganism deposited under ATCC AccessionNo. PTA-10212 (“PTA-10212”) appeared as white, wet, smeared colonieswithout visible isolated sori.

PTA-10212 was grown on solid and liquid FFM, solid KMV, KMV slush (1%),KMV broth, and MH broth to further examine growth characteristics.PTA-10212 was observed to grow rapidly on KMV and MH. See, e.g., PorterD., 1989. Phylum Labyrinthulomycota. In Margulis, L., Corliss, J. O.,Melkonian, M., Chapman, D. J. (Eds.) Handbook of Protoctista, Jones andBartlett, Boston, pp. 388-398 (KMV); Honda et al., Mycol. Res.102:439-448 (1998) (MH); and U.S. Pat. No. 5,130,242 (FFM).

The following observations were made after growth of PTA-10212 overseveral days on solid FFM media, after 72 hours growth in KMV medias,and MH broth. Sporangia were not clumped in/on any media and were verysmall (5-10 min). PTA-10212 did not demonstrate the copious tetradscharacteristic of Schizochytrium cleavage patterns. Amoeboid cellsappeared about 24 hours after transfer to fresh solid media. Theseamoeboid cells were gone after a few days and were not evident in liquidor slush media. Unlike Aurantiochytrium, described by Yokoyama, R. etal., Mycoscience 48(6): 329-341 (2007), as having the appearance of“small sandgrains on the bottom of the flask” when grown in liquidmedia, PTA-10212 did not settle at the bottom of the flask but wassuspended in both KMV and MH liquid media. The sporangia were not asdense as typical of Schizochytrium or Oligochytrium, which also haverobust ectoplasmic networks that were absent from PTA-10212. While mostspecies undergo vegetative cleavage of small sporangia or assimilativecells by the division of a larger sporangium over several hours,PTA-10212 formed dumbbell-shaped elongated assimilative cells, whichthen formed a thin isthmus that pulled apart as the ends of the dumbbellseparated. The resulting cells appeared to be small assimilative cells.Direct transformation of an amoeboid cell into the dumbbell shapedassimilative cell was not observed. Typical biflagellate zoospores wereobserved swimming but were relatively rare. PTA-10212 was non-prolific,dividing by vegetative cleavage. Direct release of zoospores was notobserved, although zoospores were observed swimming. Vegetative cellswere very small (2 μm to 5 μm).

PTA-10212 was further examined using the flow-through technique, inwhich microscopic slides were prepared by suspending a small portion ofan agar-grown colony in a drop of half-strength sea water. With thistechnique, primary sporangia were observed to be globose andapproximately 10 μm in diameter. Walls were very thin and remnants werenot observed when binary division of the protoplast was initiated.Repeated binary division produced 8-16 smaller (4-5 μm in diameter)secondary sporangia. The secondary sporangia remained quiescent forseveral hours before again releasing an amorphous protoplast. Theamorphous protoplast divided by pinching and pulling, initiallyproducing typical dumbbell-shaped intermediate stages and finallyresulting in 4-8 small globose bodies 2.5-2.8 μm in diameter. The latterrested for several minutes up to 1-2 hours, then changed shape(elongated) and turned into biflagellate zoospores, 2.3-2.5×3.7-3.9 μm.Zoospores were abundant and could be precisely measured when they cameto rest. Zoospores then rounded off and started a new cycle ofdevelopment. The zoospores were larger than Sicyoidochytrium minutum andsmaller than Ulkenia visurgensis.

PTA-10212 was further characterized based on the similarity of its 18srRNA gene to that of known species. Genomic DNA was prepared fromPTA-10212 by standard procedures. See, for example, Sambrook J. andRussell D. 2001, Molecular cloning: A laboratory manual, 3rd edition.Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Briefly:(1) 500 μL of cells were centrifuged from mid-log culture. The cellswere re-centrifuged, and all traces of liquid were removed from the cellpellet with a small-bore tip; (2) pellets were resuspended with 200 μLlysis buffer (20 mM Tris pH 8.0, 125 μg/mL Proteinase K, 50 mM NaCl, 10mM EDTA pH 8.0, 0.5% SDS); (3) cells were lysed at 50° C. for 1 hour;(4) the lysis mixture was pipetted into phase-lock gel (PLG-Eppendorf) 2mL tubes; (5) equal volume of P:C:I was added and allowed to mix for 1.5hours; (6) the tubes were centrifuged at 12,000×g for 5 minutes; (7) theaqueous phase was removed from above the gel within the PLG tube and anequal volume of chloroform was added to the aqueous phase, and mixed for30 minutes; (8) the tubes were centrifuged at 14,000×g for approximately5 minutes; (9) the top layer (aqueous phase) was pipetted away from thechloroform, and placed in a new tube; (10) 0.1 volume of 3 M NaOAC wasadded and mixed (inverted several times); (11) 2 volumes of 100% EtOHwere added and mixed (inverted several times) with genomic DNAprecipitant forming at this stage; (12) the tubes were centrifuged at 4°C. in a microcentrifuge at 14,000×g for approximately 15 minutes; (13)the liquid was gently poured off with genomic DNA remaining at thebottom of the tube; (14) the pellet was washed with 0.5 mL 70% EtOH;(15) the tubes were centrifuged at 4° C. in a microcentrifuge at14,000×g for approximately 5 minutes; (16) the EtOH was gently pouredoff and the genomic DNA pellet was dried; and (17) a suitable volume ofH₂O and RNase was added directly to the genomic DNA pellet. The PCRamplification of the 18s rRNA gene was carried out with primerspreviously described (Honda et. al., J. Euk. Micro. 46(6): 637-647(1999). The PCR conditions with chromosomal DNA template were asfollows: 0.2 μM dNTPs, 0.1 μM each primer, 8% DMSO, 200 ng chromosomalDNA, 2.5 U Herculase® II Fusion DNA Polymerase (Stratagene), andHerculase® buffer (Stratagene) in a 50 μL total volume. The PCR Protocolincluded the following steps: (1) 95° C. for 2 minutes; (2) 95° C. for35 seconds; (3) 55° C. for 35 seconds; (4) 72° C. for 1 minute and 30seconds; (5) repeat steps 2-4 for 30 cycles; (6) 72° C. for 5 minutes;and (7) hold at 4° C.

PCR amplification yielded a distinct DNA product with the expected sizeusing chromosomal template described above. The PCR product was clonedinto the vector pJET1.2/blunt (Fermentas) according to themanufacturer's instructions, and the insert sequence was determinedusing supplied standard primers.

Phylogenetic analysis places PTA-10212 within the lineage that includesThraustochytrium pachydermum and Thraustochytrium aggregatum withmoderate support. The sporangia of T. pachydermum have very thick cellwalls. T. aggregatum forms clearly visible clumps of sori that areopaque. PTA-10212 shows neither of these characteristics. The presenceof many amoeboid cells has been described in other taxa, such asUlkenia, T. gaertnerium, A. limiacinum, and S. mangrovei; however, thedescriptions associated with those taxa differ from the observedcharacteristics of the isolate. Moreover, PTA-10212 did not showphylogenetic affinity towards any of these taxa.

Table 29 shows a comparison of the 18s rRNA sequence from themicroorganism deposited under ATCC Accession No. PTA-10212 to DNAsequences in the National Center for Biotechnology Information (NCBI)electronic database. The percent identity was determined using twodifferent calculations. “Calculation #1” takes into consideration any“gaps” that occurs in the sequences, either from non-homologous regionsor partial sequence (AlignX-VectorNTI default settings). “Calculation#2” does not include calculated penalties for gaps (AlignX-VectorNTI“IDENTITY” matrix setting).

TABLE 29 Comparison of 18s rRNA Sequences % Identity % IdentityThraustochytrids Calculation #1 Calculation #2 Thraustochytriumpachydermum 85% 93% Thraustochytrium aggregatum (p) 83% 92%Thraustochytrium gaertnerium 82% 92% Ulkenia visurgensis 82% 92%Schizochytrium sp. PTA-9695 80% 92% Schizochytrium mangrovei 80% 91%Schizochytrium sp. ATCC 20888 80% 90% Aurantiochytrium limiacinum 79%90% (p): indicates partial sequence

As shown in Table 29, it was found that, in terms of % identity, the 18srRNA gene sequence from the microorganism deposited under ATCC AccessionNo. PTA-10212 is related, though not identical, to 18s rRNA genesequences available in the NCBI database. It is generally recognizedthat organisms can have closely related 18s rRNA gene sequences whilebelonging to a different genus or species.

Based on the above characterization, the isolated microorganism (ATCCAccession No. PTA-10212) is believed to represent a new Thraustochytriumspecies and is therefore also designated as Thraustochytrium sp. ATCCPTA-10212.

Taxonomic Characteristics of the Isolated Microorganism Deposited UnderATCC Accession No. PTA-10208

The microorganism deposited under ATCC Accession No. PTA-10208(“PTA-10208”) was identified as a sub-isolate (an individual cellisolated from a culture and maintained as a new separate and distinctculture) of the microorganism deposited under ATCC Accession No.PTA-9695 (“PTA-9695”), described in U.S. Pub. No. 2010/0239533 andInt'l. Pub. No. WO 2010/107415.

PTA-10208 shares taxonomic characteristics with PTA-9695. PTA-9695 wasfound to have biflagellate zoospores at discharge that swim activelyaway from the mature sporangium, wall remnants of which were clearlyvisible (in phase contrast) after spore release. PTA-9695 sporangiameasured 12.5 m to 25 μm in diameter, and zoospores were 2.5 μm to 2.8μm×4.5 μm to 4.8 μm in size. There were 8 to 24 spores per individualPTA-9695 sporangium. Settled PTA-9695 zoospores enlarged and rapidlyunderwent binary divisions leading to tetrads, octads, and finally toclusters of sporangia. Tetrad formation commenced at a very early stageprior to maturity of the sporangia. These characteristics are inagreement with the genus Schizochytrium. In terms of % identity, thePTA-9695 18s rRNA gene sequence, which is shared by PTA-10208, was foundto be closely related, though not identical, to the 18s rRNA genesequence of T. aggregatum provided in Honda, D. et al., J. Euk. Micro.46(6): 637-647 (1999). The 18s rRNA sequence published forThraustochytrium aggregatum is a partial sequence, with an approximately71 DNA nucleotide gap in the middle of the sequence. PTA-9695 isbelieved to represent a new Schizochytrium species. As such, thesub-isolate PTA-10208 is also designated as Schizochytrium sp. ATCCPTA-10208.

Example 35

Growth Characteristics of the Isolated Microorganism Deposited UnderATCC Accession No. PTA-10212

The isolated microorganism (ATCC Accession No. PTA-10212) was examinedfor growth characteristics in individual fermentation runs, as describedbelow. Typical media and cultivation conditions are shown in Table 30.

TABLE 30 PTA-10212 Vessel Media Ingredient concentration ranges Na₂SO₄g/L 31.0 0-50, 15-45, or 25-35 NaCl g/L 0.625 0-25, 0.1-10, or 0.5-5 KClg/L 1.0 0-5, 0.25-3, or 0.5-2 MgSO₄•7H₂O g/L 5.0 0-10, 2-8, or 3-6(NH₄)₂SO₄ g/L 0.44 0-10, 0.25-5, or 0.05-3 MSG•1H₂O g/L 6.0 0-10, 4-8,or 5-7 CaCl₂ g/L 0.29 0.1-5, 0.15-3, or 0.2-1 T 154 (yeast g/L 6.0 0-20,0.1-10, or 1-7 extract) KH₂PO₄ g/L 0.8 0.1-10, 0.5-5, or 0.6-1.8 Postautoclave (Metals) Citric acid mg/L 3.5 0.1-5000, 10-3000, or 3-2500FeSO₄•7H₂O mg/L 10.30 0.1-100, 1-50, or 5-25 MnCl₂•4H₂O mg/L 3.100.1-100, 1-50, or 2-25 ZnSO₄•7H₂O mg/L 3.10 0.01-100, 1-50, or 2-25CoCl₂•6H₂O mg/L 0.04 0-1, 0.001-0.1, or 0.01-0.1 Na₂MoO₄•2H₂O mg/L 0.040.001-1, 0.005-0.5, or 0.01-0.1 CuSO₄•5H₂O mg/L 2.07 0.1-100, 0.5-50, or1-25 NiSO₄•6H₂O mg/L 2.07 0.1-100, 0.5-50, or 1-25 Post autoclave(Vitamins) Thiamine mg/L 9.75 0.1-100, 1-50, or 5-25 Vitamin B12 mg/L0.16 0.01-100, 0.05-5, or 0.1-1 Ca½-panto- mg/L 2.06 0.1-100, 0.1-50, or1-10 thenate Biotin mg/L 3.21 0.1-100, 0.1-50, or 1-10 Post autoclave(Carbon) Glycerol g/L 30.0 5-150, 10-100, or 20-50 Nitrogen Feed:MSG•1H₂O g/L 17 0-150, 10-100, or 15-50 Typical cultivation conditionswould include the following: pH about 6.5 - about 9.5, about 6.5 - about8.0, or about 6.8 - about 7.8; temperature: about 15 - about 30 degreesCelsius, about 18 - about 28 degrees Celsius, or about 21 to about 23degrees Celsius; dissolved oxygen: about 0.1 - about 100% saturation,about 5 - about 50% saturation, or about 10 - about 30% saturation;and/or glucose controlled @: about 5 - about 50 g/L, about 10 - about 40g/L, or about 15 - about 35 g/L.

In carbon (glycerol) and nitrogen-fed cultures with 1000 ppm Cl⁻ at22.5° C. with 20% dissolved oxygen at pH 7.3, PTA-10212 produced a drycell weight of 26.2 g/L after 138 hours of culture in a 10 L fermentorvolume. The lipid yield was 7.9 g/L; the omega-3 yield was 5.3 g/L; theEPA yield was 3.3 g/L; and the DHA yield was 1.8 g/L. The fatty acidcontent was 30.3% by weight; the EPA content was 41.4% of fatty acidmethyl esters (FAME); and the DHA content was 26.2% of FAME. The lipidproductivity was 1.38 g/L/day, and the omega-3 productivity was 0.92g/L/day under these conditions, with 0.57 g/L/day EPA productivity and0.31 g/L/day DHA productivity.

In carbon (glycerol) and nitrogen-fed cultures with 1000 ppm CV at 22.5°C. with 20% dissolved oxygen at pH 7.3, PTA-10212 produced a dry cellweight of 38.4 g/L after 189 hours of culture in a 10 L fermentorvolume. The lipid yield was 18 g/L; the omega-3 yield was 12 g/L; theEPA yield was 5 g/L; and the DHA yield was 6.8 g/L. The fatty acidcontent was 45% by weight; the EPA content was 27.8% of FAME; and theDHA content was 37.9% of FAME. The lipid productivity was 2.3 g/L/day,and the omega-3 productivity was 1.5 g/L/day under these conditions,with 0.63 g/L/day EPA productivity and 0.86 g/L/day DHA productivity.

In carbon (glycerol) and nitrogen-fed cultures with 1000 ppm Cl⁻ at22.5° C. with 20% dissolved oxygen at pH 6.8-7.7, PTA-10212 produced adry cell weight of 13 g/L after 189 hours of culture in a 10 L fermentorvolume. The lipid yield was 5.6 g/L; the omega-3 yield was 3.5 g/L; theEPA yield was 1.55 g/L; and the DHA yield was 1.9 g/L. The fatty acidcontent was 38% by weight; the EPA content was 29.5% of FAME; and theDHA content was 36% of FAME. The lipid productivity was 0.67 g/L/day,and the omega-3 productivity was 0.4 g/L/day under these conditions,with 0.20 g/L/day EPA productivity and 0.24 g/L/day DHA productivity.

In carbon (glycerol) and nitrogen-fed cultures with 1000 ppm C1 at22.5-28.5° C. with 20% dissolved oxygen at pH 6.6-7.2, PTA-10212produced a dry cell weight of 36.7 g/L-48.7 g/L after 191 hours ofculture in a 10 L fermentor volume. The lipid yield was 15.2 g/L-25.3g/L; the omega-3 yield was 9.3 g/L-13.8 g/L; the EPA yield was 2.5g/L-3.3 g/L; and the DHA yield was 5.8 g/L-11 g/L. The fatty acidcontent was 42.4%-53% by weight; the EPA content was 9.8%-22% of FAME;and the DHA content was 38.1%-43.6% of FAME. The lipid productivity was1.9 g/L/day-3.2 g/L/day, and the omega-3 productivity was 1.2g/L/day-1.7 g/L/day under these conditions, with 0.31 g/L/day-0.41g/L/day EPA productivity and 0.72 g/L/day-1.4 g/L/day DHA productivity.

Growth Characteristics of the Isolated Microorganism Deposited UnderATCC Accession No. PTA-10208

The isolated microorganism (ATCC Accession No. PTA-10208) was examinedfor growth characteristics in individual fermentation runs, as describedbelow. Typical media and cultivation conditions are shown in Table 31.

TABLE 31 PTA-10208 Vessel Media Ingredient concentration ranges Na₂SO₄g/L 8.8 0-25, 2-20, or 3-10 NaCl g/L 0.625 0-25, 0.1-10, or 0.5-5 KClg/L 1.0 0-5, 0.25-3, or 0.5-2 MgSO₄•7H₂O g/L 5.0 0-10, 2-8, or 3-6(NH₄)₂SO₄ g/L 0.42 0-10, 0.25-5, or 0.05-3 CaCl₂ g/L 0.29 0.1-5, 0.15-3,or 0.2-1 T 154 (yeast g/L 1.0 0-20, 0.1-10, or 0.5-5 extract) KH₂PO₄ g/L1.765 0.1-10, 0.5-5, or 1-3 Post autoclave (Metals) Citric acid mg/L46.82 0.1-5000, 10-3000, or 40-2500 FeSO₄•7H₂O mg/L 10.30 0.1-100, 1-50,or 5-25 MnCl₂•4H₂O mg/L 3.10 0.1-100, 1-50, or 2-25 ZnSO₄•7H₂O mg/L 9.30.01-100, 1-50, or 2-25 CoCl₂•6H₂O mg/L 0.04 0-1, 0.001-0.1, or 0.01-0.1Na₂MoO₄•2H₂O mg/L 0.04 0.001-1, 0.005-0.5, or 0.01-0.1 CuSO₄•5H₂O mg/L2.07 0.1-100, 0.5-50, or 1-25 NiSO₄•6H₂O mg/L 2.07 0.1-100, 0.5-50, or1-25 Post autoclave (Vitamins) Thiamine mg/L 9.75 0.1-100, 1-50, or 5-25Ca½-panto- mg/L 3.33 0.1-100, 0.1-50, or 1-10 thenate Biotin mg/L 3.580.1-100, 0.1-50, or 1-10 Post autoclave (Carbon) Glucose g/L 30.0 5-150,10-100, or 20-50 Nitrogen Feed: NH₄OH mL/L 23.6 0-150, 10-100, or 15-50Typical cultivation conditions would include the following: pH about6.5 - about 8.5, about 6.5 - about 8.0, or about 7.0 - about 8.0;temperature: about 17 - about 30 degrees Celsius, about 20 - about 28degrees Celsius, or about 22 to about 24 degrees Celsius; dissolvedoxygen: about 2 - about 100% saturation, about 5 - about 50% saturation,or about 7 - about 20% saturation; and/or glucose controlled @: about5 - about 50 g/L, about 10 - about 40 g/L, or about 20 - about 35 g/L.

In carbon (glucose) and nitrogen-fed cultures with 1000 ppm Cl⁻ at 22.5°C. at pH 7.0 with 20% dissolved oxygen during the nitrogen feed and 10%dissolved oxygen thereafter, PTA-10208 produced a dry cell weight of 95g/L after 200 hours of culture in a 10 L fermentor volume. The lipidyield was 53.7 g/L; the omega-3 yield was 37 g/L; the EPA yield was 14.3g/L; and the DHA yield was 21 g/L. The fatty acid content was 57% byweight; the EPA content was 27.7% of FAME; and the DHA content was 39.1%of FAME. The lipid productivity was 6.4 g/L/day, and the omega-3productivity was 4.4 g/L/day under these conditions, with 1.7 g/L/dayEPA productivity and 2.5 g/L/day DHA productivity.

In carbon (glucose) and nitrogen-fed cultures with 1000 ppm Cl⁻ at 22.5°C. at pH 7.5 with 20% dissolved oxygen during the nitrogen feed and 10%dissolved oxygen thereafter, PTA-10208 produced a dry cell weight of 56g/L after 139 hours of culture in a 10 L fermentor volume. The lipidyield was 53 g/L; the omega-3 yield was 34 g/L; the EPA yield was 11.5g/L; and the DHA yield was 22 g/L. The fatty acid content was 58% byweight; the EPA content was 21.7% of FAME; and the DHA content was 41.7%of FAME. The lipid productivity was 9.2 g/L/day, and the omega-3productivity was 5.9 g/L/day under these conditions, with 2 g/L/day EPAproductivity and 3.8 g/L/day DHA productivity.

In carbon (glucose) and nitrogen-fed cultures with 1000 ppm Cl⁻ at 22.5°C. at pH 7.0 with 20% dissolved oxygen during the nitrogen feed and 10%dissolved oxygen thereafter, PTA-10208 produced a dry cell weight of93.8 g/L after 167 hours of culture in a 2000 L fermentor volume. Thelipid yield was 47.2 g/L; the omega-3 yield was 33.1 g/L; the EPA yieldwas 10.5 g/L; and the DHA yield was 20.4 g/L. The fatty acid content was50.6% by weight; the EPA content was 23% of FAME; and the DHA contentwas 42.6% of FAME. The lipid productivity was 6.8 g/L/day, and theomega-3 productivity was 4.7 g/L/day under these conditions, with 1.5g/L/day EPA productivity and 2.9 g/L/day DHA productivity.

In carbon (glucose) and nitrogen-fed cultures with 1000 ppm Cl⁻ at 22.5°C. at pH 7.0 with 20% dissolved oxygen during the nitrogen feed and 10%dissolved oxygen thereafter, PTA-10208 produced a dry cell weight of 105g/L after 168 hours of culture in a 2000 L fermentor volume. The lipidyield was 46.4 g/L; the omega-3 yield was 33 g/L; the EPA yield was 10.7g/L; and the DHA yield was 20.3 g/L. The fatty acid content was 43.9% byweight; the EPA content was 24% of FAME; and the DHA content was 43.7%of FAME. The lipid productivity was 6.6 g/L/day, and the omega-3productivity was 4.7 g/L/day under these conditions, with 1.5 g/L/dayEPA productivity and 2.9 g/L/day DHA productivity.

In carbon (glucose) and nitrogen-fed cultures with 1000 ppm Cl⁻ at 22.5°C. at pH 7.0 with 20% dissolved oxygen during the nitrogen feed and 10%dissolved oxygen thereafter, PTA-10208 produced a dry cell weight of64.8 g/L after 168 hours of culture in a 2000 L fermentor volume. Thelipid yield was 38.7 g/L; the omega-3 yield was 29.9 g/L; the EPA yieldwas 8.5 g/L; and the DHA yield was 16.7 g/L. The fatty acid content was59.6% by weight; the EPA content was 23% of FAME; and the DHA contentwas 42.3% of FAME. The lipid productivity was 5.53 g/L/day, and theomega-3 productivity was 3.8 g/L/day under these conditions, with 1.2g/L/day EPA productivity and 2.3 g/L/day DHA productivity.

Example 36

Fatty Acid Profiles of Microorganism Strains ATCC PTA-10208 andPTA-10212

Four samples of biomass (PTA-10208 Sample #1; PTA-10208 Sample #2;PTA-10212 Sample #1; and PTA-10212 Sample #2) were analyzed for totalcrude oil content by solvent extraction, lipid classes were determinedby high performance liquid chromatography/evaporative light scatteringdetection (HPLC/ELSD), triacylglycerol (TAG) were analyzed by HPLC/massspectrometry (HPLC/MS), and fatty acid (FA) profiles were determined bygas chromatography with flame ionization detection (GC-FID). The crudelipid content of each freeze dried biomass was determined using solventgrinding with hexane and compared to the sum of FAME (mg/g) generated bydirect transesterification, and the resultant fatty acid methyl esters(FAME) were quantified by GC/FID analysis. Fatty acids in the extractedcrude lipid were also quantified by transesterification and quantifiedusing GC/FID analysis of the resultant FAME. The weight percent of allneutral lipids (NL) and free fatty acids (FFA) were determined in theextracted crude lipid using normal phase HPLC with ELSD and atmosphericpressure chemical ionization-MS (APCI-MS) identification. The methodseparates and quantifies sterol esters (SE), TAG, free fatty acids(FFA), 1,3-diacylglycerols (1,3-DAG), sterols, 1,2-diacylglycerols(1,2-DAG), and monoacylglycerols (MAG). Results are shown in Tables 32and 33, below. It is noted that fatty acid profiles of Tables 32 and 33were obtained from samples extracted using a solvent. The fatty acidprofiles of Tables 32 and 33 are expected to be the substantially thesame if the samples were extracted using the processes of the presentinvention.

TAG and phospholipids (PL) were isolated from the crude oils extractedfrom the four samples of biomass (PTA-10208 Sample #1; PTA-10208 Sample#2; PTA-10212 Sample #1; and PTA-10212 Sample #2). TAG was isolatedusing low pressure flash chromatography and PL was isolated using solidphase extraction (SPE). The identity of each isolated fraction wasconfirmed by thin layer chromatography (TLC). The fatty acid profiles ofthe isolated TAG and PL fractions were determined following directtransesterification using GC-FID as FAME. Results are shown in Tables 34and 35, below.

The total crude oil content and fatty acid profiles of isolated lipidclasses were also determined for two additional samples of biomass frommicroorganism strain ATCC PTA-10212 (PTA-10212 Sample #3 and PTA-10212Sample #4). Crude oil was obtained from each sample by hexaneextraction, and individual lipid classes were isolated using lowpressure flash chromatography. The fatty acid profiles of the biomass,crude oil, and isolated fractions were determined using GC-FID as FAME.Results are shown in Tables 36-39, below. It is noted that fatty acidprofiles of Tables 36-39 were obtained from samples extracted using atypical hexane extraction. The fatty acid profiles of Tables 36-39 areexpected to be the substantially the same if the samples were extractedusing the processes of the present invention.

Individual lipid classes were isolated from a sample of crude oil frommicroorganism strain ATCC PTA-10212 (PTA-10212 Sample #5) previouslyextracted using the FRIOLEX® process, and the fatty acid profiles ofeach class were determined using GC-FID as FAME. Results are shown inTables 40 and 41, below. It is noted that fatty acid profiles of Tables40 and 41 were obtained from samples extracted using a FRIOLEX® process.The fatty acid profiles of Tables 40 and 41 are expected to be thesubstantially the same if the samples were extracted using the processesof the present invention.

Individual lipid classes were isolated from a sample of crude oil frommicroorganism strain ATCC PTA-10208 (PTA-10208 Sample #3) using normalHPLC with ELSD and APCI-MS identification.

Experimental Procedures

Crude Oil Extraction—

Crude oil was extracted from samples of freeze-dried biomass usingsolvent grinding. For example, approximately 3 grams of biomass wasweighed into a Swedish tube. Three ball bearings and 30 mL of hexanewere added to the Swedish tube, which was sealed with a neoprene stopperand placed in a shaker for 2 hours. The resultant slurry was filteredusing a Buchner funnel and Whatman filter paper. The filtered liquid wascollected, the solvent removed under vacuum, and the amount of remainingcrude lipid determined gravimetrically.

Fatty Acid Analysis—

The samples of biomass, extracted crude lipid, and isolated lipidclasses were analyzed for fatty acid composition as FAME. Briefly,freeze-dried biomass and isolated lipid classes were weighed directlyinto a screw cap test tubes, while samples of the crude oil weredissolved in hexane to give a concentration of approximately 2 mg/mL.Toluene, containing internal standard, and 1.5 N HCl in methanol wasadded to each tube. The tubes were vortexed, then capped and heated to100° C. for 2 hours. The tubes were allowed to cool, and saturated NaClin water was added. The tubes were vortexed again and centrifuged toallow the layers to separate. A portion of the organic layer was thenplaced in a GC vial and analyzed by GC-FID. FAME was quantified using a3-point calibration curve generated using Nu-Check-Prep GLC ReferenceStandard (NuCheck, Elysian, Minn.). Fatty acids present in the extractwere expressed as mg/g and as a weight percent. Fat content in thesamples was estimated assuming equal response to the internal standardwhen analyzed by GC-FID.

HPLC/ELSD/MS Method—

Instrument Agilent 1100 HPLC, Alltech 3300 ELSD, Agilent 1100 MSD ColumnPhenomenex Luna Silica, 250 × 4.6 mm, 5 μm particle size w/Guard ColumnMobile Phase A - 99.5% Hexanes (Omnisolv) 0.4% Isopropyl alcohol(Omnisolv) 0.1% Acetic Acid B - 99.9% Ethanol (Omnisolv, 95:5Ethanol:IPA) 0.1% Acetic Acid

Gradient Time, min. % A % B 0 100 0 5 100 0 15 85 10 20 0 100 25 0 10026 100 0 35 100 0

Column Temp. 30° C. Flow Rate 1.5 mL/min Injection Volume 5 μL ELSDDetection Temperature 35° C., Gas flow 1.2 L/min MSD Mass Range200-1200, Fragmentor 225 V Drying Gas Temperature 350° C. VaporizerTemperature 325° C. Capillary Voltage 3500 V Corona Current 10 μA

Solid Phase Extraction—

PL fractions were separated from the crude lipid by solid phaseextraction (SPE) using 2 g aminopropyl cartridges (Biotage, Uppsala,Sweden) placed in a Vac Elut apparatus (Varian Inc, Palo Alto, USA). Thecartridge was conditioned with 15 mL of hexane, and ˜60 mg of eachsample was dissolved in 1 mL CHCl₃ and applied to the cartridge. Thecolumn was washed with 15 mL of 2:1 CHCl₃:isopropyl alcohol to elute allthe neutral lipids, which was discarded. The fatty acids were theneluted with 15 mL of 2% acetic acid (HOAc) in ether, which wasdiscarded. The PL portion was eluted with 15 mL of 6:1Methanol:Chloroform, which was collected, dried under nitrogen, andweighed.

Flash Chromatography—

Flash chromatography was used to separate the lipid classes present inthe crude oil. Approximately 200 mg of crude oil dissolved in hexane wasinjected onto the head of the column. The chromatography system utilizedSilica Gel 60 (EMD Chemical, Gibbstown, N.J.) with mobile phase composedof Petroleum Ether and Ethyl Acetate at 5 mL/min (Tables 6-7) or 3mL/min (Tables 8-13). A step gradient was used to selectively elute eachlipid class from the column. The mobile phase gradient started from 100%petroleum ether and finished with 50% ethyl acetate. Fractions werecollected in 10 mL test tubes using a Gilson FC 204 large-bed fractioncollector (Gilson, Inc., Middleton, Wis.). Each tube was analyzed bythin layer chromatography (TLC) and the tubes containing individuallipid classes (as judged by single spots on TLC plate with expectedretention factor (Rf)) were pooled, concentrated to dryness, andweighed. The total fraction content was then determined gravimetrically.

TLC Analysis—

Thin layer chromatography was conducted on silica gel plates. The plateswere eluted using a solvent system consisting of petroleum ether:ethylether:acetic acid (80:20:1) and were visualized using iodine vapor. TheRf values of each spot were then compared with reported literaturevalues for each lipid class.

Analysis of TAG and PL Fractions—

The isolated TAG and PL fractions were analyzed for fatty acidcomposition as fatty acid methyl esters (FAME). The TAG fractions weredissolved in hexane to give a concentration of approximately 1-2 mg/mL.1 mL aliquots of the solutions were concentrated to dryness undernitrogen. Toluene, containing internal standard, and 1.5 N HCl inmethanol was added to each tube. The tubes were vortexed, then cappedand heated to 100° C. for 2 hours. Internal standard and HCl methanolwere added directly to the tubes containing the PL fraction and heated.The tubes were allowed to cool, and saturated NaCl in water was added.The tubes were vortexed again and centrifuged to allow the layers toseparate. A portion of the organic layer was then placed in a GC vialand analyzed by GC-FID. FAMEs were quantified using a 3-pointcalibration curve generated using Nu-Check-Prep GLC 502B ReferenceStandard (NuCheck, Elysian, Minn.). Fatty acids present in the extractwere expressed as mg/g and as a % of FAME.

Results

PTA-10208 Sample #1

The fatty acid profile of the biomass and extracted crude lipid forPTA-10208 Sample #1 was determined using GC/FID. Fatty acids in thebiomass were transesterified in situ by weighing 28.6 mg of biomassdirectly into a FAME tube, while a sample of the extracted crude lipidwas prepared by weighing 55.0 mg of crude lipid into a 50 mL volumetricflask and transferring 1 ml to a separate FAME tube. The estimated crudelipid content of the biomass was determined to be 53.2% (as SUM of FAME)using GC with FID detection, while 52.0% (wt/wt) lipid was extractedfrom the dry biomass, giving a 97.8% recovery of total lipid. The crudelipid was determined to be 91.9% fatty acids (as SUM of FAME) usingGC/FID. The major fatty acids contained in the crude lipid were C16:0(182.5 mg/g), C20:5 n-3 (186.8 mg/g), and C22:6 n-3 (423.1 mg/g).

The lipid class profile of the extracted crude lipid was determined byweighing 55.0 mg of crude lipid into a 50 mL volumetric flask andtransferring an aliquot into an HPLC vial for HPLC/ELSD/MS analysis.According to the HPLC/ELSD/MS analysis, the crude lipid contained 0.2%sterol esters (SE), 95.1% TAG, 0.4% sterols, and 0.5% 1,2-diacylglycerol(DAG). 5% of the TAG fraction included a peak that eluted directly afterthe TAG peak, but did not give a recognizable mass spectrum.

Isolated TAG from this sample as determined by flash chromatography madeup approximately 92.4% of the crude oil. PL was not detected by weightor TLC after SPE isolation. The major fatty acids (>50 mg/g) containedin the TAG were C16:0 (189 mg/g), C20:5 n-3 (197 mg/g), and C22:6 n-3(441 mg/g).

PTA-10208 Sample #2

The fatty acid profile of the biomass and extracted crude lipid forPTA-10208 Sample #2 was determined using GC/FID. Fatty acids in thebiomass were transesterified in situ by weighing 32.0 mg of biomassdirectly into a FAME tube, while a sample of the extracted crude lipidwas prepared by weighing 60.1 mg of crude lipid into a 50 mL volumetricflask and transferring 1 ml to a separate FAME tube. The estimated crudelipid content of the biomass was determined to be 52.4% (as SUM of FAME)using GC with FID detection, while 48.0% (wt/wt) lipid was extractedfrom the dry biomass, giving a 91.7% recovery of total lipid. The crudelipid was determined to be 95.3% fatty acids (as SUM of FAME) usingGC/FID. The major fatty acids contained in the crude lipid were C16:0(217.5 mg/g), C20:5 n-3 (169.3 mg/g), and C22:6 n-3 (444.1 mg/g).

The lipid class profile of the extracted crude lipid was determined byweighing 60.1 mg of crude lipid into a 50 mL volumetric flask andtransferring an aliquot into an HPLC vial for HPLC/ELSD/MS analysis.According to the HPLC/ELSD/MS analysis, the crude lipid contained 0.2%SE, 95.7% TAG, 0.3% sterols, and 0.7% 1,2-DAG. 5.1% of the TAG fractionincluded a peak that eluted directly after the TAG peak, but did notgive a recognizable mass spectrum.

Isolated TAG from this sample made up approximately 93.9% of the crudeoil. PL was not detected by weight or TLC after SPE isolation. The majorfatty acids (>50 mg/g) contained in the TAG were C16:0 (218 mg/g), C20:5n-3 (167 mg/g) and C22:6 n-3 (430 mg/g).

PTA-10208 Sample #3

A sample of crude oil from the microorganism deposited under ATCCAccession No. PTA-10208 (Sample PTA-10208 #3) was analyzed usingHPLC/ELSD/MS. A total of 98.38% of lipids were recovered, with thesterol ester (SE) fraction accounting for 0.32%, the TAG fractionaccounting for 96.13%, the 1,3-diacylglycerol (DAG) fraction accountingfor 0.22%, the 1,2-DAG fraction accounting for 0.78%, and the sterolfraction accounting for 0.93%.

PTA-10212 Sample #1

The fatty acid profile of the biomass and extracted crude lipid forPTA-10212 Sample #1 was determined using GC/FID. Fatty acids in thebiomass were transesterified in situ by weighing 27.0 mg of biomassdirectly into a FAME tube, while a sample of the extracted crude lipidwas prepared by weighing 52.5 mg of crude lipid into a 50 mL volumetricflask and transferring 1 ml to a separate FAME tube. The estimated crudelipid content of the biomass was determined to be 38.3% (as SUM of FAME)using GC with FID detection, while 36.3% (wt/wt) lipid was extractedfrom the dry biomass, giving a 94.6% recovery of total lipid. The crudelipid was determined to be 83.2% fatty acids (as SUM of FAME) usingGC/FID. The major fatty acids contained in the crude lipid were C16:0(328.5 mg/g), C20:5 n-3 (90.08 mg/g), and C22:6 n-3 (289.3 mg/g).

The lipid class profile of the extracted crude lipid was determined byweighing 52.5 mg of crude lipid into a 50 mL volumetric flask andtransferring an aliquot into an HPLC vial for HPLC/ELSD/MS analysis.According to the HPLC/ELSD/MS analysis, the crude lipid contained 0.2%SE, 64.2% TAG, 1.9% FFA, 2.8% 1,3-DAG, 1.4% sterols, 18.8% 1,2-DAG, and0.5% MAG. 3.4% of the TAG fraction included a peak that eluted directlyafter the TAG peak, but did not give a recognizable mass spectrum.

Isolated TAG from this sample made up approximately 49.8% of the crudeoil. Isolated PL made up approximately 8.1% of the crude oil. The majorfatty acids (>50 mg/g) contained in the TAG fraction are C16:0 (400mg/g), C20:5 n-3 (91 mg/g), and C22:6 n-3 (273 mg/g). The major fattyacids (>50 mg/g) contained in the PL fraction are C16:0 (98 mg/g), C20:5n-3 (33 mg/g), and C22:6 n-3 (227 mg/g).

PTA-10212 Sample #2

The fatty acid profile of the biomass and extracted crude lipidPTA-10212 Sample #2 was determined using GC/FID. Fatty acids in thebiomass were transesterified in situ by weighing 29.5 mg of biomassdirectly into a FAME tube, while a sample of the extracted crude lipidwas prepared by weighing 56.5 mg of crude lipid into a 50 mL volumetricflask and transferring 1 ml to a separate FAME tube. The estimated crudelipid content of the biomass was determined to be 40.0% (as SUM of FAME)using GC with FID detection, while 41.3% (wt/wt) lipid was extractedfrom the dry biomass, giving a 106.1% recovery of total lipid. The crudelipid was determined to be 82.8% fatty acids (as SUM of FAME) usingGC/FID. The major fatty acids contained in the crude lipid were C16:0(327.3 mg/g), C20:5 n-3 (92.5 mg/g), and C22:6 n-3 (277.6 mg/g).

The lipid class profile of the extracted crude lipid was determined byweighing 56.5 mg of crude lipid into a 50 mL volumetric flask andtransferring an aliquot into an HPLC vial for HPLC/ELSD/MS analysis.According to the HPLC/ELSD/MS analysis, the crude lipid contained 0.2%SE, 58.2% TAG, 2.3% FFA, 3.4% 1,3-DAG, 1.7% sterols, 23.4% 1,2-DAG, and0.6% MAG. 3.3% of the TAG fraction included a peak that eluted directlyafter the TAG peak, but did not give a recognizable mass spectrum.

Isolated TAG from this sample made up approximately 51.9% of the crudeoil. Isolated PL made up approximately 8.8% of the crude oil. The majorfatty acids (>50 mg/g) contained in the TAG fraction are C16:0 (402mg/g), C20:5 n-3 (92 mg/g), and C22:6 n-3 (245 mg/g). The major fattyacids (>50 mg/g) contained in the PL fraction are C16:0 (121 mg/g),C20:5 n-3 (48 mg/g), and C22:6 n-3 (246 mg/g).

TABLE 32 Fatty Acid Profiles of PTA-10208 and PTA-10212 Biomasses andExtracted Crude Lipids (mg/g) PTA- PTA- PTA- PTA- PTA- PTA- PTA- 1020810208 10208 10208 10212 10212 10212 PTA- Sample Sample #1 Sample Sample#2 Sample Sample #1 Sample 10212 #1 Crude #2 Crude #1 Crude #2 Sample #2Biomass Lipid Biomass Lipid Biomass Lipid Biomass Crude Lipid Fatty FAMEFAME FAME FAME FAME FAME FAME FAME Acid (mg/g) (mg/g) (mg/g) (mg/g)(mg/g) (mg/g) (mg/g) (mg/g) C12:0 1.47 2.43 1.80 3.14 0.99 1.90 0.871.91 C14:0 11.62 20.12 16.72 31.03 5.51 12.91 5.97 13.69 C14:1 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 C15:0 2.43 3.75 3.60 6.22 9.13 20.42 9.3920.81 C16:0 105.04 182.47 117.72 217.49 145.87 328.45 147.87 327.27C16:1 0.00 0.00 0.06 0.01 6.26 14.53 7.46 16.89 C18:0 5.37 8.96 4.778.37 6.77 15.39 6.77 15.15 C18:1 0.00 3.26 0.00 3.09 0.03 4.04 0.08 5.87n-9 C18:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 n-7 C18:2 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 n-6 C20:0 1.48 1.79 1.40 1.85 1.60 3.091.67 3.20 C18:3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 n-3 C20:1 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 n-9 C18:4 0.91 1.61 1.10 2.00 2.282.56 2.21 2.64 n-3 C20:2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 n-6C20:3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 n-6 C22:0 0.10 0.00 0.080.00 0.30 0.12 0.35 0.24 C20:4 0.81 0.45 0.67 0.41 0.00 0.00 0.00 0.00n-7 C20:4 7.22 12.23 6.84 12.18 1.19 2.26 1.31 2.32 n-6 C22:1 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 n-9 C20:4 0.63 0.52 0.00 0.46 0.00 0.000.00 0.00 n-5 C20:4 3.45 5.45 3.33 5.58 0.00 2.40 0.00 2.34 n-3 C20:30.09 0.00 0.11 0.00 0.00 0.00 0.00 0.00 n-3 C20:5 107.31 186.83 92.99169.32 40.32 90.08 43.15 92.54 n-3 C22:4 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 n-9 C24:0 0.60 0.00 0.52 0.00 2.81 6.83 2.74 6.53 C24:1 1.553.26 0.85 2.04 0.43 1.34 0.42 1.24 n-9 C22:5 9.66 15.84 10.27 17.98 2.424.68 2.32 4.21 n-6 C22:5 20.44 35.13 9.92 17.50 2.41 4.94 2.69 5.23 n-3C22:6 246.98 423.10 245.96 444.08 139.58 289.34 137.35 277.57 n-3 Sum527.15 907.18 518.71 942.75 367.89 805.29 372.63 799.68 of FAME

TABLE 33 Fatty Acid Profiles of PTA-10208 and PTA-10212 Biomasses andExtracted Crude Lipids (%) PTA- PTA- PTA- PTA- PTA- 10208 PTA- 10208PTA- 10212 PTA- 10212 10208 Sample 10208 Sample 10212 Sample 10212Sample Sample #1 Sample #2 Sample #1 Sample #2 #1 Crude #2 Crude #1Crude #2 Crude Biomass Lipid Biomass Lipid Biomass Lipid Biomass LipidFatty % % % % % % % % Acid FAME FAME FAME FAME FAME FAME FAME FAME C12:00.28 0.27 0.35 0.33 0.27 0.24 0.23 0.24 C14:0 2.20 2.22 3.22 3.29 1.501.60 1.60 1.71 C14:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C15:0 0.460.41 0.69 0.66 2.48 2.54 2.52 2.60 C16:0 19.93 20.11 22.70 23.07 39.6540.79 39.68 40.93 C16:1 0.00 0.00 0.01 0.00 1.70 1.80 2.00 2.11 C18:01.02 0.99 0.92 0.89 1.84 1.91 1.82 1.89 C18:1 0.00 0.36 0.00 0.33 0.010.50 0.02 0.73 n-9 C18:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 n-7C18:2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 n-6 C20:0 0.28 0.20 0.270.20 0.43 0.38 0.45 0.40 C18:3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00n-3 C20:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 n-9 C18:4 0.17 0.180.21 0.21 0.62 0.32 0.59 0.33 n-3 C20:2 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 n-6 C20:3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 n-6 C22:00.02 0.00 0.01 0.00 0.08 0.02 0.09 0.03 C20:4 0.15 0.05 0.13 0.04 0.000.00 0.00 0.00 n-7 C20:4 1.37 1.35 1.32 1.29 0.32 0.28 0.35 0.29 n-6C22:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 n-9 C20:4 0.12 0.06 0.000.05 0.00 0.00 0.00 0.00 n-5 C20:4 0.65 0.60 0.64 0.59 0.00 0.30 0.000.29 n-3 C20:3 0.02 0.00 0.02 0.00 0.00 0.00 0.00 0.00 n-3 C20:5 20.3620.59 17.93 17.96 10.96 11.19 11.58 11.57 n-3 C22:4 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 n-9 C24:0 0.11 0.00 0.10 0.00 0.76 0.85 0.74 0.82C24:1 0.29 0.36 0.16 0.22 0.12 0.17 0.11 0.16 n-9 C22:5 1.83 1.75 1.981.91 0.66 0.58 0.62 0.53 n-6 C22:5 3.88 3.87 1.91 1.86 0.65 0.61 0.720.65 n-3 C22:6 46.85 46.64 47.42 47.10 37.94 35.93 36.86 34.71 n-3 Sum100 100 100 100 100 100 100 100 of FAME %

TABLE 34 Fatty Acid Profiles of PTA-10208 and PTA-10212 Isolated TAGPTA- PTA- PTA- PTA- PTA- PTA- PTA- PTA- 10208 10208 10208 10208 1021210212 10212 10212 Sample Sample Sample Sample Sample Sample SampleSample #1 #1 #2 #2 #1 #1 #2 #2 Fatty FAME % FAME % FAME % FAME % Acid(mg/g) FAME (mg/g) FAME (mg/g) FAME (mg/g) FAME C12:0 2.57 0.27 3.350.36 0.00 0.00 0.00 0.00 C14:0 21.07 2.23 31.37 3.41 14.05 1.61 14.451.69 C14:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C15:0 3.89 0.41 6.170.67 23.27 2.66 23.14 2.71 C16:0 189.28 20.07 218.78 23.75 399.51 45.75402.43 47.07 C16:1 0.00 0.00 0.00 0.00 15.23 1.74 17.62 2.06 C18:0 9.210.98 8.07 0.88 22.70 2.60 23.10 2.70 C18:1 3.35 0.36 3.64 0.40 6.12 0.707.48 0.87 n-9 C18:1 0.00 0.00 0.00 0.00 <0.1 <0.1 <0.1 <0.1 n-7 C18:20.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 n-6 C20:0 1.86 0.20 1.55 0.174.76 0.55 5.32 0.62 C18:3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 n-3C20:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 n-9 C18:4 1.64 0.17 2.000.22 2.25 0.26 2.24 0.26 n-3 C20:2 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 n-6 C20:3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 n-6 C22:0 0.000.00 0.00 0.00 0.55 0.06 0.89 0.10 Unknown 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 C20:4 0.39 0.04 0.05 0.01 0.00 0.00 0.00 0.00 n-7 C20:3 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 n-3 C20:4 12.79 1.36 11.82 1.28 2.330.27 2.25 0.26 n-6 C22:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 n-9C20:4 0.39 0.04 0.07 0.01 0.00 0.00 0.00 0.00 n-5 C20:4 5.52 0.59 5.090.55 2.87 0.33 2.98 0.35 n-3 C20:5 197.14 20.90 166.68 18.10 91.17 10.4491.78 10.74 n-3 C24:0 0.00 0.00 0.00 0.00 6.93 0.79 7.36 0.86 C22:4 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 n-9 C24:1 1.08 0.11 <0.1 <0.1 0.000.00 0.00 0.00 n-9 C22:5 15.88 1.68 16.57 1.80 4.01 0.46 3.39 0.40 n-6C22:5 36.05 3.82 16.00 1.74 4.53 0.52 5.07 0.59 n-3 C22:6 440.99 46.76429.83 46.67 273.02 31.26 245.38 28.70 n-3 Sum 943.11 — 921.03 — 873.31— 854.89 — of FAME Total % — 100.00 — 100.00 — 100.00 — 100.00 FAME

TABLE 35 Fatty Acid Profiles of PTA-10212 Isolated PL PTA-10212PTA-10212 Sample #1 PTA-10212 Sample #2 PTA-10212 FAME Sample #1 FAMESample #2 Fatty Acid (mg/g) % FAME (mg/g) % FAME C12:0 0.00 0.00 0.000.00 C14:0 0.93 0.22 1.89 0.39 C14:1 0.00 0.00 0.00 0.00 C15:0 3.44 0.825.07 1.05 C16:0 98.29 23.50 120.98 25.00 C16:1 1.15 0.27 3.07 0.63 C18:03.25 0.78 3.72 0.77 C18:1 n-9 1.12 0.27 0.95 0.20 C18:1 n-7 <0.1 <0.10.02 0.003 C18:2 n-6 0.00 0.00 0.00 0.00 C20:0 <0.1 <0.1 <0.1 <0.1 C18:3n-3 0.00 0.00 0.00 0.00 C20:1 n-9 0.00 0.00 0.00 0.00 C18:4 n-3 3.710.89 3.24 0.67 C20:2 n-6 0.00 0.00 0.00 0.00 C20:3 n-6 0.00 0.00 0.000.00 C22:0 0.00 0.00 0.00 0.00 Unknown 42.33 10.12 44.71 9.24 C20:4 n-70.00 0.00 0.00 0.00 C20:3 n-3 0.00 0.00 0.00 0.00 C20:4 n-6 0.84 0.201.54 0.32 C22:1 n-9 0.00 0.00 0.00 0.00 C20:4 n-5 0.00 0.00 0.00 0.00C20:4 n-3 <0.1 <0.1 0.27 0.06 C20:5 n-3 33.39 7.98 47.91 9.90 C24:0 0.1<0.1 0.01 0.001 C22:4 n-9 0.00 0.00 0.00 0.00 C24:1 n-9 0.00 0.00 0.000.00 C22:5 n-6 3.08 0.74 3.82 0.79 C22:5 n-3 <0.1 <0.1 0.66 0.14 C22:6n-3 226.68 54.20 246.09 50.85 Sum of FAME 418.21 — 483.94 — Total % FAME— 100 — 100

PTA-10212 Sample #3

The lipid content of the biomass for PTA-10212 Sample #3 was estimatedto be 34% as the sum of FAME, and the amount of crude oil obtained aftersolvent extraction was 37% by weight, giving a 109% recovery of fatpresent in the biomass. After fractionation using flash chromatography,approximately 46% of the crude oil was isolated as TAG, 28% was isolatedas DAG. The crude oil contained 309 mg/g DHA and 264 mg/g EPA. Theisolated TAG contained 341 mg/g DHA and 274 mg/g EPA. The isolated DAGfraction contained 262 mg/g DHA and 237 mg/g EPA. The total fatty acidprofiles of the biomass, extracted crude oil, and isolated fractions areshown below in Table 36 and Table 37 calculated as mg/g and % FAME,respectively.

TABLE 36 Fatty Acid Profiles of PTA-10212 Sample #3 Biomass andExtracted Crude Lipid (mg/g) Crude Biomass Oil TAG DAG Wt. % NA 37.2%46.0% 27.9% FAME FAME FAME FAME Fatty Acid (mg/g) (mg/g) (mg/g) (mg/g)C12:0 0.0 0.0 0.0 0.0 C14:0 3.6 10.3 11.5 9.4 C14:1 0.0 0.0 0.0 0.0C15:0 4.1 10.6 9.8 6.6 C16:0 70.5 181.8 231.7 111.3 C16:1 6.7 19.1 18.717.1 C18:0 2.4 10.2 14.2 0.0 C18:1 n-9 0.0 6.7 0.0 0.0 C18:1 n-7 0.0 1.20.0 0.0 C18:2 n-6 0.0 1.8 0.0 0.0 C20:0 0.0 2.4 0.0 0.0 C18:3 n-3 0.00.0 0.0 0.0 C20:1 n-9 0.0 0.3 0.0 1.7 C18:4 n-3 1.9 3.4 3.2 4.4 C20:2n-6 0.0 0.0 0.0 0.0 C20:3 n-6 0.0 0.0 0.0 0.0 C22:0 3.3 0.0 0.0 0.0C20:4 n-7 0.0 2.1 1.5 0.0 C20:3 n-3 0.0 0.0 0.0 0.0 C20:4 n-6 6.8 17.921.4 13.8 C22:1 n-9 0.0 0.0 0.0 0.0 C20:4 n-5 0.0 1.3 1.3 0.0 C20:4 n-33.0 8.5 10.9 6.4 C20:5 n-3 102.0 263.6 274.2 237.4 C24:0 0.0 1.7 3.9 0.0C22:4 n-9 0.0 0.0 0.0 0.0 C24:1 n-9 0.0 0.0 4.2 0.0 C22:5 n-6 3.2 8.310.7 6.1 C22:5 n-3 3.8 10.4 15.1 6.6 C22:6 n-3 131.2 309.4 341.1 261.9Sum of FAME 342.4 871.1 973.2 682.6

TABLE 37 Fatty Acid Profiles of PTA-10212 Sample #3 Biomass andExtracted Crude Lipid (%) Crude Biomass Oil TAG DAG Wt. % NA 37.2% 46.0%27.9% FAME FAME FAME FAME Fatty Acid (mg/g) (mg/g) (mg/g) (mg/g) C12:00.0 0.0 0.0 0.0 C14:0 1.1 1.2 1.2 1.4 C14:1 0.0 0.0 0.0 0.0 C15:0 1.21.2 1.0 1.0 C16:0 20.6 20.9 23.8 16.3 C16:1 2.0 2.2 1.9 2.5 C18:0 0.71.2 1.5 0.0 C18:1 n-9 0.0 0.8 0.0 0.0 C18:1 n-7 0.0 0.1 0.0 0.0 C18:2n-6 0.0 0.2 0.0 0.0 C20:0 0.0 0.3 0.0 0.0 C18:3 n-3 0.0 0.0 0.0 0.0C20:1 n-9 0.0 0.0 0.0 0.2 C18:4 n-3 0.6 0.4 0.3 0.6 C20:2 n-6 0.0 0.00.0 0.0 C20:3 n-6 0.0 0.0 0.0 0.0 C22:0 1.0 0.0 0.0 0.0 C20:4 n-7 0.00.2 0.2 0.0 C20:3 n-3 0.0 0.0 0.0 0.0 C20:4 n-6 2.0 2.1 2.2 2.0 C22:1n-9 0.0 0.0 0.0 0.0 C20:4 n-5 0.0 0.1 0.1 0.0 C20:4 n-3 0.9 1.0 1.1 0.9C20:5 n-3 29.8 30.3 28.2 34.8 C24:0 0.0 0.2 0.4 0.0 C22:4 n-9 0.0 0.00.0 0.0 C24:1 n-9 0.0 0.0 0.4 0.0 C22:5 n-6 0.9 1.0 1.1 0.9 C22:5 n-31.1 1.2 1.6 1.0 C22:6 n-3 38.3 35.5 35.1 38.4 Total % FAME 100.0 100.0100.0 100.0

PTA-10212 Sample #4

PTA-10212 Sample #4 contained approximately 23% lipid determined as thesum of FAME, of which 107% was recovered using hexane extraction. Afterfractionation using flash chromatography, approximately 42% of the crudeoil was isolated as TAG, 18% was isolated as DAG. The crude oilcontained 275 mg/g DHA and 209 mg/g EPA. The isolated TAG contained 296mg/g DHA and 205 mg/g EPA. The isolated DAG fraction contained 245 mg/gDHA and 219 mg/g EPA. The total fatty acid profiles of the biomass,extracted crude oil, and isolated fractions are shown below in Table 38(mg/g) and Table 39 (% FAME).

TABLE 38 Fatty Acid Profiles of PTA-10212 Sample #4 Biomass andExtracted Crude Lipid (mg/g) Crude Biomass Oil TAG DAG Wt. % NA 24.7%42.2% 18.4% FAME FAME FAME FAME Fatty Acid (mg/g) (mg/g) (mg/g) (mg/g)C12:0 0.0 0.0 2.1 2.4 C14:0 2.0 8.3 9.8 9.6 C14:1 0.0 0.0 0.0 0.0 C15:04.8 16.8 0.4 0.9 C16:0 63.3 210.6 285.7 138.0 C16:1 1.6 6.7 7.4 7.5C18:0 2.8 12.2 19.9 4.6 C18:1 n-9 0.0 3.7 0.7 1.1 C18:1 n-7 0.0 0.0 0.31.2 C18:2 n-6 0.0 0.0 0.0 0.0 C20:0 0.0 3.3 6.0 1.5 C18:3 n-3 0.0 0.00.0 0.0 C20:1 n-9 0.0 0.0 0.7 1.2 C18:4 n-3 1.4 3.8 3.6 5.0 C20:2 n-60.0 0.0 0.0 0.0 C20:3 n-6 0.0 0.0 0.4 0.0 C22:0 1.5 0.0 1.9 0.0 C20:4n-7 0.0 0.0 0.9 0.6 C20:3 n-3 0.0 0.0 0.0 0.0 C20:4 n-6 2.5 10.1 13.010.3 C22:1 n-9 0.0 0.0 0.0 0.0 C20:4 n-5 0.0 0.0 0.8 0.7 C20:4 n-3 1.46.3 8.6 6.0 C20:5 n-3 57.6 209.1 205.4 219.0 C24:0 0.0 2.6 0.8 0.0 C22:4n-9 0.1 0.0 0.0 0.0 C24:1 n-9 0.0 0.0 1.1 0.5 C22:5 n-6 1.4 6.1 7.9 4.5C22:5 n-3 4.0 15.8 20.8 12.9 C22:6 n-3 87.7 275.0 296.4 244.8 Sum ofFAME 232.2 790.1 894.8 672.4

TABLE 39 Fatty Acid Profiles of PTA-10212 Sample #4 Biomass andExtracted Crude Lipid (%) Crude Biomass Oil TAG DAG Wt. % NA 24.7% 42.2%18.4% FAME FAME FAME FAME Fatty Acid (mg/g) (mg/g) (mg/g) (mg/g) C12:00.0 0.0 0.2 0.4 C14:0 0.9 1.0 1.1 1.4 C14:1 0.0 0.0 0.0 0.0 C15:0 2.12.1 0.0 0.1 C16:0 27.3 26.7 31.9 20.5 C16:1 0.7 0.8 0.8 1.1 C18:0 1.21.5 2.2 0.7 C18:1 n-9 0.0 0.5 0.1 0.2 C18:1 n-7 0.0 0.0 0.0 0.2 C18:2n-6 0.0 0.0 0.0 0.0 C20:0 0.0 0.4 0.7 0.2 C18:3 n-3 0.0 0.0 0.0 0.0C20:1 n-9 0.0 0.0 0.1 0.2 C18:4 n-3 0.6 0.5 0.4 0.7 C20:2 n-6 0.0 0.00.0 0.0 C20:3 n-6 0.0 0.0 0.0 0.0 C22:0 0.6 0.0 0.2 0.0 C20:4 n-7 0.00.0 0.1 0.1 C20:3 n-3 0.0 0.0 0.0 0.0 C20:4 n-6 1.1 1.3 1.5 1.5 C22:1n-9 0.0 0.0 0.0 0.0 C20:4 n-5 0.0 0.0 0.1 0.1 C20:4 n-3 0.6 0.8 1.0 0.9C20:5 n-3 24.8 26.5 23.0 32.6 C24:0 0.0 0.3 0.1 0.0 C22:4 n-9 0.0 0.00.0 0.0 C24:1 n-9 0.0 0.0 0.1 0.1 C22:5 n-6 0.6 0.8 0.9 0.7 C22:5 n-31.7 2.0 2.3 1.9 C22:6 n-3 37.8 34.8 33.1 36.4 Total % FAME 100.0 100.0100.0 100.0

PTA-10212 Sample #5

A sample of crude oil was extracted from a biomass of PTA-10212 usingthe FRIOLEX® process (GEA Westfalia Separator UK Ltd., Milton Keynes,England) to yield microbial oil PTA-10212 Sample #5. Individual lipidclasses were isolated from PTA-10212 Sample #5 using low pressure flashchromatography, and the weight percent of each class was determined. Thefatty acid profile of each class was determined using GC-FID.

Briefly, the sample was prepared by dissolving 240 mg of crude oil in600 μL of hexane and applying to the head of the column. Afterfractionation of the sample using flash chromatography, the combinedweights of all the fractions was 240 mg giving a 100% recovery. Thesterol ester fraction accounted for 0.9%, the TAG fraction accounted for42.6%, the free fatty acid (FFA) fraction accounted for 1.3%, the sterolfraction accounted for 2.2%, and the DAG fraction accounted for 41.6%.The total fatty acid profiles of the FRIOLEX® crude oil and isolatedfractions are shown below in Table 40 and Table 41 calculated as mg/gand % FAME, respectively.

TABLE 40 Fatty Acid Profiles of PTA-10212 Sample #5 Crude Oil (mg/g)Crude Oil TAG DAG Wt. % NA 42.6% 41.6% FAME FAME FAME Fatty Acid (mg/g)(mg/g) (mg/g) C12:0 0 0.7 1.0 C14:0 7.7 7.7 8.5 C14:1 0 0.0 0.0 C15:010.3 11.7 9.3 C16:0 179.3 217.7 134.6 C16:1 18.1 16.3 25.9 C18:0 8.113.2 2.3 C18:1 n-9 4.7 8.4 0.7 C18:1 n-7 0 1.8 1.0 C18:2 n-6 1.8 3.3 0.7C20:0 1.9 3.6 0.2 C18:3 n-3 0 0.0 0.0 C20:1 n-9 0 0.7 1.0 C18:4 n-3 3.12.8 3.8 C20:2 n-6 0 0.0 0.0 C20:3 n-6 0 0.6 0.4 C22:0 0 1.5 0.0 C20:4n-7 0 1.0 0.7 C20:3 n-3 0 0.0 0.0 C20:4 n-6 12.7 16.1 13.6 C22:1 n-9 00.0 0.0 C20:4 n-5 0 1.5 0.8 C20:4 n-3 6.5 9.3 6.4 C20:5 n-3 213.3 223.7252.8 C24:0 2.3 4.4 0.6 C22:4 n-9 0 1.9 0.9 C24:1 n-9 0 0.0 0.0 C22:5n-6 7.9 9.5 8.3 C22:5 n-3 13 20.6 9.7 C22:6 n-3 305.6 327.4 353.8 Sum ofFAME 796.6 905.3 837.4

TABLE 41 Fatty Acid Profiles of PTA-10212 Sample #5 Crude Oil (%) CrudeOil TAG DAG Fatty Acid % FAME % FAME % FAME C12:0 0 0.1 0.1 C14:0 1 0.91.0 C14:1 0 0.0 0.0 C15:0 1.3 1.3 1.1 C16:0 22.5 24.0 16.1 C16:1 2.3 1.83.1 C18:0 1 1.5 0.3 C18:1 n-9 0.6 0.9 0.1 C18:1 n-7 0 0.2 0.1 C18:2 n-60.2 0.4 0.1 C20:0 0.2 0.4 0.0 C18:3 n-3 0 0.0 0.0 C20:1 n-9 0 0.1 0.1C18:4 n-3 0.4 0.3 0.5 C20:2 n-6 0 0.0 0.0 C20:3 n-6 0 0.1 0.0 C22:0 00.2 0.0 C20:4 n-7 0 0.1 0.1 C20:3 n-3 0 0.0 0.0 C20:4 n-6 1.6 1.8 1.6C22:1 n-9 0 0.0 0.0 C20:4 n-5 0 0.2 0.1 C20:4 n-3 0.8 1.0 0.8 C20:5 n-326.8 24.7 30.2 C24:0 0.3 0.5 0.1 C22:4 n-9 0 0.2 0.1 C24:1 n-9 0 0.0 0.0C22:5 n-6 1 1.1 1.0 C22:5 n-3 1.6 2.3 1.2 C22:6 n-3 38.4 36.2 42.3 Total% FAME 100 100 100

Example 37

Crude oils were further processed via refining, bleaching, anddeodorizing to obtain refined oils. The refined oils were diluted withhigh oleic sunflower oil to obtain final oils with a DHA content ofapproximately 400 mg/g. Individual lipid classes were isolated and thefatty acid profiles of each class were determined using GC-FID as FAME.

PTA-10208 Final Oils

The fatty acid profiles for PTA-10208 Final Oils #1-5 are summarized inTables 42-43, including profiles associated within the isolated TAGfraction (Tables 44-45) and the isolated sterols/DAG fraction (Tables46-47).

Individual lipid classes in the final oils were also determined usingflash chromatography (Table 48) and normal HPLC with ELSD and APCI-MSconfirmation (Table 49).

TABLE 42 Fatty Acid Profiles of PTA-10208 Final Oils (mg/g) PTA-10208PTA-10208 PTA-10208 PTA-10208 PTA-10208 Final Oil #1 Final Oil #2 FinalOil #3 Final Oil #4 Final Oil #5 FAME FAME FAME FAME FAME Fatty Acid(mg/g) (mg/g) (mg/g) (mg/g) (mg/g) C12:0 2.5 2.4 2.8 2.7 2.7 C14:0 16.114.9 21.0 18.4 17.5 C14:1 0.0 0.0 0.0 0.0 0.0 C15:0 3.8 3.6 4.4 3.9 3.9C16:0 192.1 179.1 193.1 184.3 194.6 C16:1 0.4 0.5 0.5 0.5 0.5 C17:0 0.60.5 0.9 0.8 2.1 C18:0 12.8 13.9 11.5 12.3 12.9 C18:1 n-9 23.5 82.0 25.726.0 29.5 C18:1 n-7 0.2 0.7 0.1 0.1 0.1 C18:2 n-6 3.7 8.1 4.0 4.1 4.3C20:0 4.3 4.1 3.7 4.0 4.0 C18:3 n-3 0.0 0.0 0.0 0.0 0.0 C20:1 n-9 <0.10.1 <0.1 <0.1 <0.1 C18:4 n-3 2.4 2.5 2.8 2.7 2.8 C20:2 n-6 0.0 0.0 0.00.0 0.0 C20:3 n-6 0.2 0.1 0.1 0.1 0.1 C22:0 1.2 1.8 1.0 1.1 1.1 C20:4n-7 1.7 1.6 1.7 1.8 1.6 C20:3 n-3 0.0 0.0 0.0 0.0 0.0 C20:4 n-6 12.912.1 13.5 13.5 13.3 C22:1 n-9 0.0 0.0 0.0 0.0 0.0 C20:4 n-5 1.6 1.4 1.51.7 1.5 C20:4 n-3 6.0 5.7 6.0 6.0 6.1 C20:5 n-3 173.8 163.3 196.4 209.6197.9 C24:0 1.4 1.6 1.3 1.3 1.0 C22:4 \n-9 0.0 0.0 0.0 0.0 0.0 C24:1 n-93.4 3.2 2.3 2.6 2.3 C22:5 n-6 14.9 14.0 14.4 13.0 12.9 C22:5 n-3 43.941.3 32.8 40.3 36.9 C22:6 n-3 394.8 373.7 373.2 374.3 364.2 Sum of FAME918.1 932.2 914.7 925.1 914.1

TABLE 43 Fatty Acid Profiles of PTA-10208 Final Oils (%) PTA-10208PTA-10208 PTA-10208 PTA-10208 PTA-10208 Final Oil #1 Final Oil #2 FinalOil #3 Final Oil #4 Final Oil #5 Fatty Acid % FAME % FAME % FAME % FAME% FAME C12:0 0.3 0.3 0.3 0.3 0.3 C14:0 1.8 1.6 2.3 2.0 1.9 C14:1 0.0 0.00.0 0.0 0.0 C15:0 0.4 0.4 0.5 0.4 0.4 C16:0 20.9 19.2 21.1 19.9 21.3C16:1 <0.1 <0.1 <0.1 <0.1 0.1 C17:0 0.1 0.1 0.1 0.1 0.2 C18:0 1.4 1.51.3 1.3 1.4 C18:1 n-9 2.6 8.8 2.8 2.8 3.2 C18:1 n-7 <0.1 0.1 <0.1 <0.1<0.1 C18:2 n-6 0.4 0.9 0.4 0.4 0.5 C20:0 0.5 0.4 0.4 0.4 0.4 C18:3 n-30.0 0.0 0.0 0.0 0.0 C20:1 n-9 <0.1 <0.1 <0.1 <0.1 <0.1 C18:4 n-3 0.3 0.30.3 0.3 0.3 C20:2 n-6 0.0 0.0 0.0 0.0 0.0 C20:3 n-6 <0.1 <0.1 <0.1 <0.1<0.1 C22:0 0.1 0.2 0.1 0.1 0.1 C20:4 n-7 0.2 0.2 0.2 0.2 0.2 C20:3 n-30.0 0.0 0.0 0.0 0.0 C20:4 n-6 1.4 1.3 1.5 1.5 1.5 C22:1 n-9 0.0 0.0 0.00.0 0.0 C20:4 \n-5 0.2 0.2 0.2 0.2 0.2 C20:4 \n-3 0.7 0.6 0.7 0.7 0.7C20:5 n-3 18.9 17.5 21.5 22.7 21.6 C24:0 0.1 0.2 0.1 0.1 0.1 C22:4 n-90.0 0.0 0.0 0.0 0.0 C24:1 n-9 0.4 0.3 0.2 0.3 0.2 C22:5 n-6 1.6 1.5 1.61.4 1.4 C22:5 n-3 4.8 4.4 3.6 4.4 4.0 C22:6 n-3 43.0 40.1 40.8 40.5 39.9

TABLE 44 Isolated TAG Fatty Acid Profiles: PTA-10208 Final Oils (mg/g)PTA-10208 PTA-10208 PTA-10208 PTA-10208 PTA-10208 Final Oil #1 Final Oil#2 Final Oil #3 Final Oil #4 Final Oil #5 FAME FAME FAME FAME FAME FattyAcid (mg/g) (mg/g) (mg/g) (mg/g) (mg/g) C12:0 2.5 2.3 2.7 2.5 2.6 C14:016.3 15.1 21.3 18.6 18.1 C14:1 0.0 0.0 0.0 0.0 0.0 C15:0 3.9 3.6 4.4 4.04.0 C16:0 194.2 181.9 196.1 186.1 199.8 C16:1 0.4 0.4 0.6 0.5 0.7 C17:00.6 0.5 0.9 0.8 0.8 C18:0 12.9 14.2 11.7 12.5 13.2 C18:1 n-9 24.3 84.026.8 26.1 34.0 C18:1 n-7 0.1 0.7 0.1 0.1 0.3 C18:2 n-6 3.2 7.7 3.4 3.54.0 C20:0 4.4 4.2 3.8 4.0 4.2 C18:3 n-3 0.0 0.0 0.0 0.0 0.0 C20:1 n-9<0.1 0.2 <0.1 <0.1 0.1 C18:4 n-3 2.5 2.4 2.8 2.6 2.7 C20:2 n-6 0.0 0.00.0 0.0 0.0 C20:3 n-6 0.2 0.2 0.1 0.1 0.1 C22:0 1.2 1.9 1.0 1.1 1.1C20:4 n-7 1.7 1.6 1.8 1.8 1.7 C20:3 n-3 0.0 0.0 0.0 0.0 0.0 C20:4 n-613.2 12.3 13.8 13.7 13.8 C22:1 n-9 0.0 0.0 0.0 0.0 0.0 C20:4 n-5 1.6 1.51.6 1.7 1.5 C20:4 n-3 6.1 5.7 6.1 5.9 6.2 C20:5 n-3 176.0 166.1 199.0211.2 204.2 C24:0 1.2 1.3 1.0 1.1 1.2 C22:4 n-9 0.0 0.0 0.0 0.0 0.0C24:1 n-9 3.3 3.2 2.2 2.5 2.4 C22:5 n-6 15.0 14.2 14.7 13.2 13.5 C22:5n-3 44.4 42.0 33.3 40.5 38.3 C22:6 n-3 397.9 378.4 376.4 375.5 375.5 Sumof FAME 926.9 945.7 925.5 929.6 944.1

TABLE 45 Isolated TAG Fatty Acid Profiles: PTA-10208 Final Oils (%)PTA-10208 PTA-10208 PTA-10208 PTA-10208 PTA-10208 Final Oil #1 Final Oil#2 Final Oil #3 Final Oil #4 Final Oil #5 Fatty Acid % FAME % FAME %FAME % FAME % FAME C12:0 0.3 0.2 0.3 0.3 0.3 C14:0 1.8 1.6 0.3 0.3 0.3C14:1 0.0 0.0 0.0 0.0 0.0 C15:0 0.4 0.4 0.5 0.4 0.4 C16:0 20.9 19.2 21.220.0 21.2 C16:1 <0.1 <0.1 0.1 0.1 0.1 C17:0 0.1 0.1 0.1 0.1 0.1 C18:01.4 1.5 1.3 1.3 1.4 C18:1 n-9 2.6 8.9 2.9 2.8 3.6 C18:1 n-7 <0.1 0.1<0.1 <0.1 <0.1 C18:2 n-6 0.3 0.8 0.4 0.4 0.4 C20:0 0.5 0.4 0.4 0.4 0.4C18:3 n-3 0.0 0.0 0.0 0.0 0.0 C20:1 n-9 <0.1 <0.1 <0.1 <0.1 <0.1 C18:4n-3 0.3 0.3 0.3 0.3 0.3 C20:2 n-6 0.0 0.0 0.0 0.0 0.0 C20:3 n-6 <0.1<0.1 <0.1 <0.1 <0.1 C22:0 0.1 0.2 0.1 0.1 0.1 C20:4 n-7 0.2 0.2 0.2 0.20.2 C20:3 n-3 0.0 0.0 0.0 0.0 0.0 C20:4 n-6 1.4 1.3 1.5 1.5 1.5 C22:1n-9 0.0 0.0 0.0 0.0 0.0 C20:4 n-5 0.2 0.2 0.2 0.2 0.2 C20:4 n-3 0.7 0.60.7 0.6 0.7 C20:5 n-3 19.0 17.6 21.5 22.7 21.6 C24:0 0.1 0.1 0.1 0.1 0.1C22:4 n-9 0.0 0.0 0.0 0.0 0.0 C24:1 n-9 0.4 0.3 0.2 0.3 0.3 C22:5 n-61.6 1.5 1.6 1.4 1.4 C22:5 n-3 4.8 4.4 3.6 4.4 4.1 C22:6 n-3 42.9 40.040.7 40.4 39.8

TABLE 46 Isolated Sterols/DAG Fatty Acid Profiles: PTA-10208 Final Oils(mg/g) PTA-10208 PTA-10208 PTA-10208 PTA-10208 PTA-10208 Final Oil #1Final Oil #2 Final Oil #3 Final Oil #4 Final Oil #5 FAME FAME FAME FAMEFAME Fatty Acid (mg/g) (mg/g) (mg/g) (mg/g) (mg/g) C12:0 1.9 2.1 2.9 2.11.9 C14:0 9.9 9.5 9.7 10.3 8.0 C14:1 0.0 0.0 0.0 0.0 0.0 C15:0 2.4 2.32.2 2.3 2.0 C16:0 132.6 128.6 110.1 116.8 106.4 C16:1 0.2 0.3 <0.1 0.30.4 C17:0 0.3 0.2 0.3 0.3 0.3 C18:0 7.3 8.1 6.4 6.8 6.1 C18:1 n-9 15.055.1 47.4 19.0 30.1 C18:1 n-7 0.4 0.7 0.1 <0.1 0.2 C18:2 n-6 13.1 16.721.6 13.5 18.4 C20:0 2.0 2.1 1.2 1.8 1.4 C18:3 n-3 0.0 0.0 0.0 0.0 0.0C20:1 n-9 <0.1 <0.1 <0.1 <0.1 <0.1 C18:4 n-3 2.3 2.4 2.4 2.4 2.0 C20:2n-6 0.0 0.0 0.0 0.0 0.0 C20:3 n-6 <0.1 <0.1 <0.1 <0.1 <0.1 C22:0 0.6 1.00.5 0.6 0.5 C20:4 n-7 0.8 0.9 2.1 0.9 0.7 C20:3 n-3 0.0 0.0 0.0 0.0 0.0C20:4 n-6 5.7 5.8 4.8 6.1 4.5 C22:1 n-9 0.0 0.0 0.0 0.0 0.0 C20:4 n-5<0.1 <0.1 <0.1 0.6 <0.1 C20:4 n-3 2.7 2.7 2.1 2.7 2.0 C20:5 n-3 92.994.5 91.9 111.6 84.8 C24:0 1.2 1.3 1.1 1.1 1.3 C22:4 n-9 0.0 0.0 0.0 0.00.0 C24:1 n-9 1.9 2.0 1.2 1.5 1.2 C22:5 n-6 7.8 8.0 6.7 7.0 5.5 C22:5n-3 22.2 22.9 13.9 20.7 14.2 C22:6 n-3 246.3 252.7 223.5 240.3 196.3 Sumof FAME 569.3 619.8 552.1 568.7 488.2

TABLE 47 Isolated Sterols/DAG Fatty Acid Profiles: PTA-10208 Final Oils(%) PTA-10208 PTA-10208 PTA-10208 PTA-10208 PTA-10208 Final Oil #1 FinalOil #2 Final Oil #3 Final Oil #4 Final Oil #5 Fatty Acid % FAME % FAME %FAME % FAME % FAME C12:0 0.3 0.3 0.5 0.4 0.4 C14:0 1.7 1.5 1.8 1.8 1.6C14:1 0.0 0.0 0.0 0.0 0.0 C15:0 0.4 0.4 0.4 0.4 0.4 C16:0 23.3 20.8 19.920.5 21.8 C16:1 <0.1 <0.1 <0.1 <0.1 0.1 C17:0 0.0 0.0 0.1 0.1 0.1 C18:01.3 1.3 1.2 1.2 1.2 C18:1 n-9 2.6 8.9 8.6 3.3 6.2 C18:1 n-7 0.1 0.1 <0.1<0.1 <0.1 C18:2 n-6 2.3 2.7 3.9 2.4 3.8 C20:0 0.4 0.3 0.2 0.3 0.3 C18:3n-3 0.0 0.0 0.0 0.0 0.0 C20:1 n-9 <0.1 <0.1 <0.1 <0.1 <0.1 C18:4 n-3 0.40.4 0.4 0.4 0.4 C20:2 n-6 0.0 0.0 0.0 0.0 0.0 C20:3 n-6 <0.1 <0.1 <0.1<0.1 <0.1 C22:0 0.1 0.2 0.1 0.1 0.1 C20:4 n-7 0.1 0.1 0.4 0.2 0.1 C20:3n-3 0.0 0.0 0.0 0.0 0.0 C20:4 n-6 1.0 0.9 0.9 1.1 0.9 C22:1 n-9 0.0 0.00.0 0.0 0.0 C20:4 n-5 <0.1 <0.1 <0.1 0.1 <0.1 C20:4 n-3 0.5 0.4 0.4 0.50.4 C20:5 n-3 16.3 15.2 16.6 19.6 17.4 C24:0 0.2 0.2 0.2 0.2 0.3 C22:4n-9 0.0 0.0 0.0 0.0 0.0 C24:1 n-9 0.3 0.3 0.2 0.3 0.2 C22:5 n-6 1.4 1.31.2 1.2 1.1 C22:5 n-3 3.9 3.7 2.5 3.6 2.9 C22:6 n-3 43.3 40.8 40.5 42.340.2

TABLE 48 Lipid class separation by flash chromatography (wt %) LipidClass PTA-10208 PTA-10208 PTA-10208 PTA-10208 PTA-10208 Separation FinalOil #1 Final Oil #2 Final Oil #3 Final Oil #4 Final Oil #5 TAG 93.4 95.494.0 95.7 95.1 Sterols/DAG 3.1 2.9 2.6 3.0 2.9 Recovery (%) 96.5 98.396.6 98.7 98.0

TABLE 49 Lipid class separation by HPLC-ELSD (wt %) Sterol Esters TAGFFA Sterols 1,3-DAG 1,2-DAG MAG Total PTA-10208 0.4 90.8 ND 0.8 0.5 0.5N.D. 93.0 Final Oil #1 PTA-10208 0.4 88.5 ND 0.6 0.6 0.6 N.D. 90.7 FinalOil #2 PTA-10208 0.3 89.4 ND 0.8 0.6 0.5 N.D. 91.6 Final Oil #3PTA-10208 0.3 88.0 ND 0.8 0.5 0.5 N.D. 90.1 Final Oil #4 PTA-10208 0.386.3 ND 0.7 0.8 0.5 N.D. 88.6 Final Oil #5 PTA-10208 0.36 100.76 ND 0.840.54 0.61 N.D. 103.11 Final Oil #6 ND = Not Detected

PTA-10212 Final Oil

DHA was present in a PTA-10212 Final Oil at 41.63% and 366.9 mg/g, whileEPA was present at 16.52%. Individual fatty acid profiles weredetermined and are summarized in Table 50,

TABLE 50 Fatty Acid Profiles of PTA-10212 Final Oil (% FAME) Fatty Acid% FAME C6:0 ND C7:0 ND C8:0 ND C9:0 ND C10:0 ND C11:0 ND C12:0 ND C13:0ND C14:0 0.84 C14:1 ND C15:0 1.33 C16:0 27.09  C16:1 1.03 C17:0 0.34C17:1 ND C18:0 1.26 C18:1 n-9 2.14 C18:1 n-7 0.18 C19:0 ND C18:2 n-60.58 C20:0 0.32 C18:3 n-3 ND C20:1 n-9 ND C18:3 n-6 ND C20:2 n-6 0.26C20:3 n-6 ND C22:0 0.14 C20:3 n-3 ND C20:4 n-6 1.34 C22:1 n-9 ND C23:0ND C20:5 n-3 16.53  C24:0 0.53 C24:1 n-9 ND C22:5 n-6 1.50 C22:5 n-31.30 C22:6 n-3 41.63  Unknown 0.87 ND = Not Detected

Example 38

A two-day old inoculum flask of the isolated microorganisms depositedunder ATCC Accession Nos. PTA-10208 and 10212 was prepared as a carbonand nitrogen-fed culture in media according to Tables 30 and 31.

Mutagenesis was carried out according to following procedure:

A sterile T-2 day old flask, approximately 50 ml, was poured into asterile 40 ml glass homogenizer. The culture received 50 plunges in thehomogenizer. The culture was pipeted out and filtered through a sterile50 micron mesh filter, which was placed in a 50 ml sterile tube (themesh was used as a means of retaining the larger clumps of colonieswhile letting the smaller clusters and single cells pass through the 50micron mesh.). The entire concentrated macerate was collected in asterile 50 ml tube. The macerated culture was vortexed and dilutions atlevels up to 1:100 fold were made. The diluted macerate samples werevortexed prior to adding 200 μl of inoculum to a media agar plate,100×15 mm, containing 4-5 glass beads (3 mm glass beads). Each plate wasgently agitated in an effort to have the beads spread the inoculumevenly around the plate. Beads were dumped off of plates and plates wereleft to sit with covers on for approximately 5 minutes to dry. Lights inboth the sterile hood and adjoining areas were turned off as theprocedure was performed in dim light. There was minimal light availableto be able to run the procedure but only indirect and dim.

Five replicate plates were placed on the floor of the XL crosslinker(Spectronics Corporation, New York) with the lids off while the sampleswere irradiated. The crosslinker delivered power in terms of microjoulesand a level was sought that achieved a 90%-95% Kill. Five replicatecontrol plates were inoculated with un-mutagenized cells using the sameprotocol. These cell counts were used to calculate the % Kill. Once theirradiation was finished the plates were taken out, the lids werereplaced, and the plates were wrapped in parafilm followed by a wrap inaluminum foil. It was imperative that the plates grew for the first weekin the dark so that they were not able to repair the damaged genes.

Plates were placed in a 22.5° C. room for about 10 days prior tocounting the colonies. When final counts were made, individual colonieswere picked with a sterile inoculating loop and re-streaked on new mediaplates. Each colony was plated on an individual plate. As plates grewdense a sample was taken, using a inoculating loop, and inoculated intoa sterile 250 ml shake flask containing 50 ml of media. This flask wasplaced on a shaker at 200 rpm in a 22.5° C. room. On T=7 days the shakeflask culture was harvested into a 50 ml sterile tube. The pH was takenand the sample was centrifuged to collect the biomass pellet. Eachsample was rinsed and re-suspended in a 50:50 mixture of isopropylalcohol and distilled water prior to being re-centrifuged. The collectedpellet was freeze dried, weighed, and a FAME analysis was performed. Thedata in Tables 51 and 52 represents mutants produced with the aboveprocess from strains PTA-10208 and PTA-10212, respectively.

TABLE 51 PTA-10208 Mutants control Mutant 1 Mutant 2 Mutant 3 FattyAcids PTA-10208 PTA-10209 PTA-10210 PTA-10211 % 08:0 0.00 0.00 0.00 0.00% 09:0 0.00 0.00 0.00 0.00 % 10:0 0.00 0.00 0.00 0.00 % 11:0 0.00 0.000.00 0.00 % 11:1 0.00 0.00 0.00 0.00 % 12:0 0.11 0.10 0.22 0.19 % 12:10.00 0.00 0.00 0.00 % 13:0 0.19 0.19 0.15 0.16 % 13:1 0.00 0.00 0.000.00 % 14:0 1.94 1.82 2.98 2.59 % 14:1 0.00 0.00 0.00 0.00 % 15:1 2.662.22 1.76 1.66 % 16:0 24.87 24.97 23.71 25.01 % 16:1 0.20 0.25 0.07 0.07% 16:2 0.00 0.00 0.00 0.00 % 16:3 0.00 0.00 0.00 0.00 % 17:0 1.49 1.210.62 0.66 % 18:0 1.13 1.14 0.91 1.01 % 18:1 n-9 0.07 0.07 0.06 0.06 %18:1 n-7 0.00 0.00 0.00 0.00 % 18:2 0.00 0.00 0.00 0.00 % 18:3 n-6 0.000.00 0.05 0.04 % 18:3 n-3 0.09 0.08 0.17 0.14 % 18:4 n-3 0.00 0.00 0.000.00 % 20:0 0.31 0.33 0.24 0.30 % 20:1 n-9 0.00 0.04 0.00 0.00 % 20:20.00 0.00 0.05 0.00 % 20:3 n-9 0.00 0.00 0.00 0.00 % 20:3 n-6 0.12 0.130.08 0.04 % 20:3 n-3 0.42 0.42 0.08 0.06 % 20:4 ARA 0.68 0.67 1.44 1.11% 20:5 n-3 6.56 6.47 11.99 9.87 EPA % 22:0 0.07 0.07 0.06 0.07 % 22:10.00 0.00 0.00 0.00 % 22:2 0.11 0.09 0.10 0.08 % 22:3 0.00 0.00 0.000.00 % 22:4 n-6 0.00 0.00 0.00 0.00 % 22:5 n-6 2.32 2.36 2.36 2.36 %22:5 n-3 0.48 0.66 0.66 0.52 % 22:6 n-3 51.58 52.27 48.17 49.35 DHA %24:0 0.00 0.00 0.00 0.00 % 24:1 0.00 0.00 0.00 0.00 % Fat 47.87 49.4166.00 63.12 % Unknown 4.61 4.45 4.07 4.64

TABLE 52 PTA-10212 Mutants Control Mutant 1 Mutant 2 Mutant 3 FattyAcids PTA-10212 PTA-10213 PTA-10214 PTA-10215 % 08:0 0.00 0.00 0.00 0.00% 09:0 0.00 0.00 0.00 0.00 % 10:0 0.00 0.00 0.00 0.00 % 11:0 0.00 0.000.00 0.00 % 11:1 0.00 0.00 0.00 0.00 % 12:0 0.00 0.00 0.00 0.00 % 12:10.00 0.00 0.00 0.00 % 13:0 0.00 0.00 0.21 0.20 % 13:1 0.00 0.00 0.000.00 % 14:0 0.68 0.77 0.62 0.97 % 14:1 0.00 0.00 0.00 0.00 % 15:1 0.000.00 0.00 0.00 % 16:0 17.36 19.94 15.27 23.61 % 16:1 1.45 2.33 1.40 2.57% 16:2 0.00 0.00 0.00 0.00 % 16:3 0.00 0.00 0.00 0.00 % 17:0 0.20 0.210.18 0.27 % 18:0 0.78 0.82 0.79 0.81 % 18:1 n-9 0.00 0.00 0.00 0.00 %18:1 n-7 0.18 0.27 0.20 0.19 % 18:2 0.00 0.00 0.00 0.00 % 18:3 n-6 0.000.00 0.00 0.00 % 18:3 n-3 0.00 0.00 0.00 0.00 % 18:4 n-3 0.00 0.00 0.000.00 % 20:0 0.00 0.00 0.00 0.00 % 20:1 n-9 0.00 0.00 0.00 0.00 % 20:20.00 0.00 0.00 0.00 % 20:3 n-9 0.00 0.00 0.00 0.00 % 20:3 n-6 0.00 0.000.00 0.00 % 20:3 n-3 0.90 0.77 0.99 0.66 % 20:4 ARA 1.43 1.32 1.65 0.72% 20:5 n-3 13.33 14.93 14.14 8.54 EPA % 22:0 0.00 0.00 0.00 0.00 % 22:10.00 0.00 0.00 0.00 % 22:2 0.00 0.00 0.00 0.00 % 22:3 0.00 0.00 0.000.00 % 22:4 n-6 0.00 0.00 0.00 0.00 % 22:5 n-6 2.39 1.95 2.59 2.18 %22:5 n-3 0.73 0.79 0.80 0.68 % 22:6 n-3 59.18 54.31 59.89 56.39 DHA %24:0 0.00 0.00 0.00 0.00 % 24:1 0.00 0.00 0.00 0.00 % Fat 45.69 38.0842.88 48.48 % Unknown 1.38 1.58 1.27 2.19

Example 39

Two cell broths (approximately 13.3 kg) containing microbial cells(Schizochytrium) were heated to 60° C. in a 20 liter fermentor. Thefermentor had two 6-blade Rushton impellers having a diameter of 15 cm.The top impeller was positioned at the 12 liter mark and the bottomimpeller was positioned 10 cm below the top impeller. The first cellbroth was continuously agitated at 307 centimeters/second. The secondcell broth was continuously agitated at 464 centimeters/second. Enzymes(i.e., Alcalase 2.4 L FG 0.5%) were added to the cell biomass to lysethe cells and form an emulsified lysed cell composition. The emulsifiedlysed cell composition was first treated with a base (NaOH, 250 kg of50% w/w solution) until the pH of the lysed cell composition was from10.4 to 10.6. Next, a salt (solid NaCl, in an amount of 2%, by weight,of the lysed cell composition) was added to the lysed cell composition.The lysed cell composition was then heated to a temperature of 90° C.and held at that temperature level for 20 hours. A sample of each cellbroth was taken and the pH was adjusted to 8.0 and placed in 50 ml testtubes. The test tubes were centrifuged and the oil extraction data wasmeasured. The oil extraction data is provided in Table 53.

TABLE 53 Results from extraction testing in 50 mL tubes at pH 8.0. Wetbroth tested for Mass of Oil % yield % yield Extraction (g) Recovered(g) (oil/broth) (oil/solids)* 307 centimeters/second 49.990 3.881 7.7627.81 50.814 2.747 5.41 19.36 50.772 2.418 4.76 17.05 464centimeters/second 51.154 7.067 13.81 49.13 51.092 7.055 13.81 49.1150.132 6.606 13.18 46.86 *based on solids content of untreatedpasteurized broth

The data provided in Table 53 demonstrates that the higher agitationspeed resulted in a greater mass of oil recovered, a greater % yield ofoil from the broth, and a greater % yield of oil from the solids contentof the untreated pasteurized broth.

CONCLUSION

All of the various embodiments or options described herein can becombined in any and all variations. While the invention has beenparticularly shown and described with reference to some embodimentsthereof, it will be understood by those skilled in the art that theyhave been presented by way of example only, and not limitation, andvarious changes in form and details can be made therein withoutdeparting from the spirit and scope of the invention. Thus, the breadthand scope of the present invention should not be limited by any of theabove described exemplary embodiments, but should be defined only inaccordance with the following claims and their equivalents.

All documents cited herein, including journal articles or abstracts,published or corresponding U.S. or foreign patent applications, issuedor foreign patents, or any other documents, are each entirelyincorporated by reference herein, including all data, tables, figures,and text presented in the cited documents.

What is claimed is:
 1. A process for obtaining a lipid from a microbial cell composition, comprising: raising the pH of the cell composition to 8 or above; and separating a lipid from the cell composition, wherein the lipid contains less than 5% by weight of an organic solvent.
 2. The process of claim 1, wherein the raising the pH lyses the cell composition.
 3. The process of claim 1, wherein the raising the pH demulsifies the cell composition.
 4. The process of any of claims 1-3, further comprising adding a salt to the cell composition to demulsify the cell composition.
 5. The process of claim 4, wherein the adding a salt is performed after the raising the pH.
 6. The process of any of claims 1-5, further comprising heating the cell composition to demulsify the cell composition.
 7. The process of claim 6, wherein the heating is performed after the raising the pH.
 8. The process of any of claims 1-7, further comprising raising the pH of the cell composition a second time to demulsify the cell composition.
 9. The process of claim 8, wherein the raising the pH a second time is performed after the adding a salt or the heating.
 10. A process for obtaining a lipid from a cell, comprising: lysing a cell to form a lysed cell composition; raising the pH of the lysed cell composition to 8 or above to demulsify the cell composition; adding a salt to the lysed cell composition to demulsify the cell composition; and separating a lipid from the demulsified cell composition, wherein the lipid contains less than 5% by weight of an organic solvent.
 11. A process for obtaining a lipid from a cell composition, comprising: raising the pH of a cell composition to 8 or above to lyse the cell composition and demulsify the cell composition; adding a salt to the cell composition; and separating a lipid from the demulsified cell composition, wherein the lipid contains less than 5% by weight of an organic solvent.
 12. The process of claim 10 or 11, further comprising heating the lysed cell composition to demulsify the cell composition.
 13. The process of claim 12, wherein the heating is performed after the adding a salt.
 14. The process of any of claims 1-13, further comprising agitating the lysed cell composition to demulsify the cell composition.
 15. The process of any of claims 10-14, further comprising raising a pH of the lysed cell composition to demulsify the cell composition.
 16. The process of claim 15, wherein the raising the pH of the lysed cell composition is performed after the adding a salt or the heating.
 17. The process of any of claims 1-16, wherein raising the pH comprises adding a base.
 18. The process of any of claims 1-17, wherein the base has a pK_(b) of 1 to
 12. 19. The process of any of claims 14-18, wherein the process comprises agitating the lysed cell composition by stirring, mixing, blending, shaking, vibrating, or a combination thereof.
 20. The process of any of claims 1-19, wherein the lysing comprises mechanical treatment, physical treatment, chemical treatment, enzymatic treatment, or a combination thereof.
 21. The process of claim 20, wherein the mechanical treatment is homogenization.
 22. The process of any of claims 4-21, wherein the salt is added in an amount of 0.1% to 20% by weight of the lysed cell composition.
 23. The process of any of claims 4-22, wherein the salt is selected from the group consisting of: alkali metal salts, alkali earth metal salts, sulfate salts, and combinations thereof.
 24. The process of any of claims 1-23, wherein the separating comprises centrifuging.
 25. The process of any of claims 1-24, wherein the process provides a lipid comprising at least 50% by weight triglyceride.
 26. The process of any of claims 1-25, wherein the process provides a lipid having an anisidine value of 26 or less.
 27. The process of any of claims 10-26, wherein the cell is a microbial cell.
 28. The process of any of claims 1-9 and 27, further comprising concentrating a fermentation broth comprising the microbial cell.
 29. The process of any of claims 10-26, wherein the cell is an oilseed.
 30. The process of claim 29, wherein the oilseed is selected from the group consisting of sunflower seeds, canola seeds, rapeseeds, linseeds, castor oil seeds, coriander seeds, calendula seeds, and genetically modified variants thereof.
 31. The process of any of claims 1-30, further comprising washing the cell or cell composition.
 32. The process of any of claims 1-31, further comprising pasteurizing the cell or cell composition.
 33. The process of any of claims 1-32, further comprising concentrating the lysed cell composition.
 34. The process of any of claims 1-33, further comprising refining the lipid.
 35. The process of claim 34, wherein the refining is selected from the group consisting of: refining, degumming, acid treatment, alkali treatment, cooling, heating, bleaching, deodorizing, deacidification, and combinations thereof.
 36. The process of any of claims 1-35, further comprising harvesting the lipid, wherein the harvesting comprises pumping the lipid without agitation.
 37. A process for obtaining a lipid from a cell, comprising: lysing a cell to form a lysed cell composition; agitating the cell composition to demulsify the cell composition; and separating a lipid from the demulsified cell composition, wherein the lipid contains less than 5% by weight of an organic solvent.
 38. A process of claim 37, wherein the agitating comprises agitating the cell composition with an impeller having a tip speed of 350 centimeters per second to 900 centimeters per second.
 39. A lipid obtained by the process of any of claims 1-38.
 40. The lipid of claim 39, wherein the lipid comprises one or more polyunsaturated fatty acids.
 41. The lipid of any of claims 39-40, wherein the lipid comprises at least 35% by weight docosahexaenoic acid.
 42. The lipid of any of claims 39-41, wherein the lipid has an overall aroma intensity of 3 or less.
 43. The lipid of any of claims 39-41, wherein the lipid has an overall aromatic intensity of 2 or less.
 44. The lipid of any of claims 39-43, wherein the lipid has an anisidine value of 26 or less.
 45. The lipid of any of claims 39-44, wherein the lipid has a phosphorus content of 100 ppm or less.
 46. The lipid of any of claims 39-45, wherein the lipid has a peroxide value of 5 or less.
 47. The lipid of claim 39, comprising at least 20% by weight eicosapentaenoic acid and less than 5% by weight each of arachidonic acid, docosapentaenoic acid n-6, oleic acid, linoleic acid, linolenic acid, eicosenoic acid, erucic acid, and stearidonic acid.
 48. The lipid of claim 39, comprising a triacylglycerol fraction of at least 10% by weight, wherein at least 12% by weight of the fatty acids in the triacylglycerol fraction is eicosapentaenoic acid, wherein at least 25% by weight of the fatty acids in the triacylglycerol fraction is docosahexaenoic acid, and wherein less than 5% by weight of the fatty acids in the triacylglycerol fraction is arachidonic acid.
 49. The lipid of claim 47 or 48, wherein the lipid has a peroxide value of 5 or less.
 50. The lipid of any of claims 39-49, wherein the lipid is a crude oil.
 51. A crude microbial lipid having an anisidine value of 26 or less, a peroxide value of 5 or less, a phosphorus content of 100 ppm or less, and less than 5% by weight of an organic solvent.
 52. An extracted microbial lipid comprising a triglyceride fraction of at least 70% by weight, wherein the docosahexaenoic acid content of the triglyceride fraction is at least 50% by weight, wherein the docosapentaenoic acid n-6 content of the triglyceride fraction is from at least 0.5% by weight to 6% by weight, and wherein the oil has an anisidine value of 26 or less.
 53. An extracted microbial lipid comprising a triglyceride fraction of at least 70% by weight, wherein the docosahexaenoic acid content of the triglyceride fraction is at least 40% by weight, wherein the docosapentaenoic acid n-6 content of the triglyceride fraction is from at least 0.5% by weight to 6% by weight, wherein the ratio of docosahexaenoic acid to docosapentaenoic acid n-6 is greater than 6:1, and wherein the oil has an anisidine value of 26 or less.
 54. An extracted microbial lipid comprising a triglyceride fraction of at least about 70% by weight, wherein the docosahexaenoic acid content of the triglyceride fraction is at least 60% by weight and wherein the oil has an anisidine value of 26 or less.
 55. The extracted microbial lipid of any of claims 52-54, wherein the oil has a peroxide value of 5 or less.
 56. The extracted microbial lipid of any of claims 52-55, wherein the oil has a phosphorus content of 100 ppm or less.
 57. The extracted microbial lipid of any of claims 52-56, wherein the lipid is a crude oil.
 58. A process for obtaining a lipid, the process comprising refining a crude lipid of any of claims 50, 51, or
 57. 59. The process of claim 58, wherein the refining is selected from the group consisting of: caustic refining, degumming, acid treatment, alkali treatment, bleaching, deodorizing, deacidification, and combinations thereof. 